Enhanced alkyl ester containing oil compositions and methods of making and using the same

ABSTRACT

Vegetable oil compositions, as an example, corn oil, having an elevated lower alkyl ester content above about 7% weight percent of the total weight of the oil composition, and uses thereof are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to POT Application No.PCT/US2017/034262, filed on 24 May 2017, which application isincorporated herein by reference in its entirety.

BACKGROUND

Ethanol can be produced from grain-based feedstocks (e.g., corn,sorghum/milo, barley, wheat, soybeans, etc.), from sugar (e.g., sugarcane, sugar beets, etc.), or from biomass (e.g., lignocellulosicfeedstocks, such as switchgrass, corn cobs and stover, wood, or otherplant material).

In a conventional ethanol plant, corn is used as a feedstock and ethanolis produced from starch contained within the corn. Corn kernels arecleaned and milled to prepare starch-containing material for processing.Corn kernels can also be fractionated to separate the starch-containingmaterial (e.g., endosperm) from other matter (such as fiber and germ).The starch-containing material is slurried with water and liquefied tofacilitate saccharification, where the starch is converted into sugar(e.g., glucose), and fermentation, where the sugar is converted by anethanologen (e.g., yeast) into ethanol. The fermentation product isbeer, which comprises a liquid component, including ethanol, water, andsoluble components, and a solids component, including unfermentedparticulate matter (among other things). The fermentation product issent to a distillation system where the fermentation product isdistilled and dehydrated into ethanol. The residual matter (e.g., wholestillage) comprises water, soluble components, oil, and unfermentedsolids (e.g., the solids component of the beer with substantially allethanol removed, which can be dried into dried distillers grains (DOG)and sold, for example, as an animal feed product). Other co-products(e.g., syrup and oil contained in the syrup), can also be recovered fromthe whole stillage. Water removed from the fermentation product indistillation can be treated for re-use at the plant.

Various processes for recovering oil from a fermentation product arecurrently known in the art. Such processes, however, can be expensive,inefficient or even dangerous.

Conventional processes for recovering oil from a fermentation productcan sacrifice oil quality such that the oil contains a high level offree fatty acids. The presence of a high level of free fatty acids canhamper the production of end products. Processes for producing ethanol,such as the process set forth in WO 2004/081193, produce fermentationbyproducts which contain increased levels of oils while maintaining alow level of free fatty acids.

SUMMARY

The disclosure provides an oil composition comprising vegetable oilcomprising a lower alkyl ester (methyl, ethyl, propyl or butyl ester, orany combination thereof) content that is greater than 7%, e.g., a loweralkyl ester content that is greater than 18%, w/w based on the totalweight of the oil composition and optionally one or more of: an iodinevalue of not greater than 125 and/or a combined moisture and insolublecontent of no greater than 1.5% w/w based on the total weight of thecomposition; and also optionally a further component selected from thegroup consisting of: a lutein content of at least 50 mcg/g, acis-lutein/zeaxanthin content of at least 10 mcg/g, analpha-cryptoxanthin content of at least 5 mcg/g, a beta-cryptoxanthincontent of at least 5 mcg/g, an alpha-carotene content of at least 0.5mcg/g, and a cis-beta-carotene content of at least 0.1 mcg/g. In oneembodiment, the vegetable oil comprises a free fatty acid content of nogreater than 5% w/w based on the total weight of the oil composition. Inone embodiment, the vegetable oil comprises at least one fatty acidselected from the group consisting of C16 palmitic, 018 stearic, 018-1oleic, 018-2 linoleic, and 018-3 linolenic. In one embodiment, the oilcomposition further comprises an unsaponifiables content of no greaterthan 3% w/w based on the total weight of the composition. In oneembodiment, the oil composition further comprises an unsaponifiablescontent of no greater than 2.5% w/w based on the total weight of thecomposition. In one embodiment, the further component comprises a luteincontent of at least 50 mcg/g, a zeaxanthin content of at least 30 mcg/g,a cis-lutein/zeaxanthin content of at least 10 mcg/g, analpha-cryptoxanthin content of at least 5 mcg/g, a beta-cryptoxanthincontent of at least 5 mcg/g, an alpha-carotene content of at least 0.5mcg/g, a beta-carotene content of at least 1 mcg/g, a cis-beta-carotenecontent of at least 0.1 mcg/g, an alpha-tocopherol content of at least50 mcg/g, a beta-tocopherol content of at least 2 mcg/g, agamma-tocopherol content of at least 300 mcg/g, a delta-tocopherolcontent of at least 15 mcg/g, an alpha-tocotrienol content of at least50 mcg/g, a beta-tocotrienol content of at least 5 mcg/g, agamma-tocotrienol content of at least 80 mcg/g, a delta-tocotrienolcontent of at least 5 mcg/g, or any combination thereof. In oneembodiment, the lower alkyl ester content is greater than about 20% w/win the total weight of the oil composition. In one embodiment, the loweralkyl ester content is greater than about 30% w/w in the total weight ofthe oil composition. In one embodiment, the lower alkyl ester content isgreater than about 40% w/w in the total weight of the oil composition.In one embodiment, the lower alkyl ester content is greater than about50% w/w in the total weight of the oil composition. In one embodiment,the lower alkyl ester content is greater than about 60% w/w in the totalweight of the oil composition. In one embodiment, the lower alkyl estercontent is greater than about 70% w/w in the total weight of the oilcomposition. In one embodiment, the lower alkyl ester content is greaterthan about 80% w/w in the total weight of the oil composition. In oneembodiment, the oil composition is a fuel composition. In oneembodiment, the oil composition is a fuel additive. In one embodiment,the oil composition is an asphalt rejuvenator. In one embodiment, theoil composition is an asphalt performance enhancer.

Recycled asphalt in pavement and shingles is often very stiff andviscous which can cause premature cracking due to lack of durability aswell as loss of workability in its use. Recycled asphalt can berejuvenated by reducing the viscosity, softening, and increasing thedurability of asphalt mixtures by addition of vegetable oil enhancedwith fatty acid esters such as ethyl esters (FAEE). Additionally, such amaterial can be used to modify the grade of various performance grade(PG) asphalts in order to improve the low temperature properties. Highethyl ester containing vegetable oil is shown here to rejuvenaterecycled asphalt and improve low temperature properties of virginasphalt in the aforementioned ways better than vegetable oil with alower ethyl ester content.

Further provided is a method to alter one or more properties of asphalt,e.g., recycled asphalt, virgin asphalt or performance-grade asphalt. Themethod includes in one embodiment combining recycled asphalt,performance-grade asphalt, or recycled asphalt and virgin asphalt, andan amount of a vegetable oil composition effective to alter at least oneproperty of the asphalt, thereby forming an asphalt mix composition (ifaggregates are present, e.g., from the recycled asphalt), or an asphaltbinder blend composition (if aggregates are absent), wherein thevegetable oil has a lower alkyl ester content that is greater than 7%,e.g., ester content that is greater than 18%, w/w based on the totalweight of the oil composition. Optionally the vegetable oil has aniodine value of not greater than 125 and/or a combined moisture andinsoluble content of no greater than 1.5% w/w based on the total weightof the composition; and also optionally a further component selectedfrom the group consisting of: a lutein content of at least 50 mcg/g, acis-lutein/zeaxanthin content of at least 10 mcg/g, analpha-cryptoxanthin content of at least 5 mcg/g, a beta-cryptoxanthincontent of at least 5 mcg/g, an alpha-carotene content of at least 0.5mcg/g, and a cis-beta-carotene content of at least 0.1 mcg/g. In oneembodiment, the vegetable oil has a free fatty acid content of nogreater than 5% w/w based on the total weight of the composition. In oneembodiment, the free fatty acid content of the oil composition comprisesat least one fatly acid selected from the group consisting of C16palmitic, C18 stearic, 018-1 oleic, C18-2 linoleic, and C18-3 linolenic.In one embodiment, the oil composition further comprises anunsaponifiables content of no greater than 3% w/w based on the totalweight of the composition. In one embodiment, the oil compositionfurther comprises an unsaponifiables content of no greater than 2.5% w/wbased on the total weight of the composition. In one embodiment, thelower alkyl ester content is greater than about 30% w/w in the totalweight of the oil composition. In one embodiment, the lower alkyl estercontent is greater than about 50% w/w in the total weight of the oilcomposition. In one embodiment, the lower alkyl ester content is greaterthan about 20% w/w in the total weight of the oil composition. In oneembodiment, the lower alkyl ester content is greater than about 60% w/win the total weight of the oil composition. In one embodiment, the oilcomposition is about 0.5% w/w to about 50% w/w the total weight of thebitumen without aggregates (referred to as an asphalt bindercomposition), or a combined weight of the bitumen and the oilcomposition (an asphalt binder blend). In one embodiment, the oilcomposition is about 1% w/w to about 50% w/w the total weight of theasphalt binder composition, or a combined weight of the asphalt bindercomposition and the oil composition. In one embodiment, the oilcomposition is about 1% w/w to about 25% w/w the total weight of theasphalt binder composition or a combined weight of the asphalt bindercomposition and the oil composition. In one embodiment, the oilcomposition is about 1% w/w to about 10% w/w the total weight of theasphalt binder composition, or a combined weight of the asphalt bindercomposition and the oil composition.

Also provided is an asphalt binder blend composition comprising abitumen composition (without aggregates; an asphalt binder composition)and a vegetable oil composition having a lower alkyl ester content thatis greater than 7%, e.g., an ethyl ester content that is greater than18%, w/w based on the total weight of the oil composition; andoptionally an iodine value of not greater than 125 and/or a combinedmoisture and insoluble content of no greater than 1.5% w/w based on thetotal weight of the composition; and also optionally a further componentselected from the group consisting of: a lutein content of at least 50mcg/g, a cis-lutein/zeaxanthin content of at least 10 mcg/g, analpha-cryptoxanthin content of at least 5 mcg/g, a beta-cryptoxanthincontent of at least 5 mcg/g, an alpha-carotene content of at least 0.5mcg/g, and a cis-beta-carotene content of at least 0.1 mcg/g. In oneembodiment, the vegetable oil is about 0.5 wt % to about 25 wt % of theweight of the asphalt binder composition (bitumen without aggregates),or a combined weight of the asphalt binder composition and the oilcomposition. In one embodiment, the vegetable oil is about 4 wt % toabout 12 wt % of weight of the bitumen composition (without aggregates),or a combined weight of the asphalt binder composition and the oilcomposition (asphalt binder blend). In one embodiment, the vegetable oilis about 5 wt % to 10 wt % of the weight of the of the asphalt bindercomposition (bitumen without aggregates), or a combined weight of theasphalt binder composition and the oil composition. In one embodiment,the vegetable oil is about 0.5 wt % to about 50 wt % of the weight ofthe asphalt binder composition, or a combined weight of the asphaltbinder composition and the oil composition. In one embodiment, thevegetable oil is about 1 wt % to about 25 wt % of the weight of the ofthe asphalt hinder composition (bitumen without aggregates), or acombined weight of the asphalt binder composition and the oilcomposition. In one embodiment, the lower alkyl ester content is greaterthan about 18% and up to about 80% w/w in the total weight of the oilcomposition. In one embodiment, the lower alkyl ester content is greaterthan about 20% up to about 60% w/w in the total weight of the oilcomposition. In one embodiment, the lower alkyl ester content is greaterthan about 30% and up to about 50% w/w in the total weight of the oilcomposition. In one embodiment, the vegetable oil has a free fatty acidcontent of no greater than 5% w/w based on the total weight of the oilcomposition. In one embodiment, the asphalt comprises recycled asphalt.In one embodiment, the asphalt comprises virgin asphalt. In oneembodiment, the asphalt comprises performance grade asphalt. In oneembodiment, the asphalt comprises recycled asphalt. In one embodiment,the asphalt hinder composition comprises an emulsion, e.g., which alsoincludes water and an emulsifier.

Further provided is a pavement or paving composition (asphalt mix)comprising aggregate, e.g., virgin aggregate, and from about 1.0% toabout 10.0% of an asphalt binder composition and a vegetable oilcomposition having: a lower alkyl ester content that is greater thanabout 7%, such as greater than 18%, w/w based on the total weight of thecomposition; and optionally an iodine value of not greater than 125and/or a combined moisture and insoluble content of no greater than 1.5%w/w based on the total weight of the composition; and also optionally afurther component selected from the group consisting of: a luteincontent of at least 50 mcg/g, a cis-lutein/zeaxanthin content of atleast 10 mcg/g, an alpha-cryptoxanthin content of at least 5 mcg/g, abeta-cryptoxanthin content of at least 5 mcg/g, an alpha-carotenecontent of at least 0.5 mcg/g, and a cis-beta-carotene content of atleast 0.1 mcg/g. Methods of making a paving composition are alsoprovided. In one embodiment, the lower alkyl ester content is greaterthan about 20% and up to about 60% w/w in the total weight of the oilcomposition. In one embodiment, the lower alkyl ester content is greaterthan about 30% up to about 50% w/w in the total weight of the oilcomposition. In one embodiment, the lower alkyl ester content is greaterthan about 30% and up to about 70% w/w in the total weight of the oilcomposition. In one embodiment, the vegetable oil has a free fatty acidcontent of no greater than 5% w/w based on the total weight in the oilcomposition.

In addition, an asphalt mix composition is provided comprising: bitumen,aggregate and a vegetable oil composition having a lower alkyl estercontent that is greater than 7%, e.g., greater than about 18%, w/w basedon the total weight of the oil composition; and optionally an iodinevalue of not greater than 125 and/or a combined moisture and insolublecontent of no greater than 1.5% w/w based on the total weight of thecomposition; and also optionally a further component selected from thegroup consisting of: a lutein content of at least 50 mcg/g, acis-lutein/zeaxanthin content of at least 10 mcg/g, analpha-cryptoxanthin content of at least 5 mcg/g, a beta-cryptoxanthincontent of at least 5 mcg/g, an alpha-carotene content of at least 0.5mcg/g, and a cis-beta-carotene content of at least 0.1 mcg/g. In oneembodiment, the lower alkyl ester content in the vegetable oil isgreater than about 20% and up to about 60% w/w in the total weight ofthe oil composition. In one embodiment, the lower alkyl ester content inthe vegetable oil is greater than about 30% up to about 70% w/w in thetotal weight of the oil composition. In one embodiment, the lower alkylester content in the vegetable oil is greater than about 40% and up toabout 80% w/w in the total weight of the oil composition. In oneembodiment, the vegetable oil has a free fatty acid content of nogreater than 5% w/w based on the total weight of the composition. In oneembodiment, the aggregate comprises a plurality of solids comprisingsand, gravel, crushed stone, crushed concrete, crushed glass, industrialslag, or any combination thereof. In one embodiment, the asphalt mix isa combination of virgin asphalt and recycled asphalt. In one embodiment,the vegetable oil is about 0.5 wt % to about 25 wt % of the weight ofthe asphalt binder composition (bitumen without aggregates), or acombined weight of the asphalt binder composition and the oilcomposition. In one embodiment, the vegetable oil is about 4 wt to about12 wt % of weight of the asphalt binder composition (bitumen withoutaggregates), or a combined weight of the asphalt binder composition andthe oil composition. In one embodiment, the vegetable oil is about 5 wt% to 10 wt % of the weight of the asphalt binder composition or acombined weight of the asphalt binder composition and the oilcomposition. In one embodiment, the vegetable oil is about 0.5 wt % toabout 50 wt % of the weight of asphalt binder composition, or a combinedweight of the asphalt binder composition and the oil composition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic block flow diagram of a process for producingethanol from corn.

FIG. 2 is a schematic flow diagram of a process for producing ethanolfrom corn.

FIG. 3 shows the effect of pH on the fatty acid content of the oilcomposition.

FIG. 4 shows an exemplary process flow diagram.

FIGS. 5A-E show various exemplary flow diagrams for providing the oilcomposition and the distillers dried grains.

FIG. 6 shows that conventional ethanol fermentation including aliquefaction step prior to fermentation decreases the ethyl estercontent of the extracted oil post fermentation compared to a controlcorn composition (BPX). N=5 fermentations for both conventional and BPX.

FIG. 7 shows that addition of lipase at the beginning of BPXfermentation increases the level of FAEE in corn oil extracted at theend of fermentation. The various enzyme doses of control (0.0%), 0.04%,0.4%, and 4.0% are based upon lipase weight added to weight of corn fatavailable in the fermenter. Each dose was performed in duplicate.

FIG. 8 shows that reduction of viscosity as a function of ethyl estercontent in corn oil. The dynamic viscosity of corn oil at 25° C. isreduced as ethyl ester concentration is increased. Data was obtainedwith a Brookfield viscometer.

FIG. 9 shows that effect of corn oil rejuvenators with 3% and 100% ethylesters (EE) content on ΔT_(c) of aged asphalt. An increase in ΔT_(c) isfavorable and is a measure of the relative durability of the asphalt.Values were obtained from the bending beam rheometer test (AASHTO T313).

FIG. 10 shows performance grade tests demonstrating modification of a64-22 asphalt to a 58-28 and 52-34 with 4 and 7 percent inclusion ofdistiller's corn oil (DCO), respectively.

FIG. 11 shows that DCO at 4 percent inclusion significantly increasesthe cracking resistance of the asphalt mixture containing 50% RAP. Testswere carried out by overlay tester (TxDOT Tex-248-F).

FIG. 12 shows the effect on rutting by inclusion of 4 percent DCO in a50% RAP mixture compared to 50% RAP control. Line shown on graphindicates the maximal rutting specification of 12.5 mm over 10,000 wheelpasses. Tests were carried out by Hamburg Wheel Track (AASHTO 1-324).

DETAILED DESCRIPTION

This disclosure relates to a vegetable oil, e.g., corn oil, compositionwith enhanced lower alkyl ester content and a method for producing thesame, as well as the use of vegetable oil, e.g., corn oil, compositions,for example, to enhance the properties of performance grade orrejuvenated asphalt.

It is to be understood that this invention is not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of this invention will be limited only by theappended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “analkali metal ion” includes a plurality of alkali metal ions.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. As used herein the followingterms have the following meanings.

As used herein, the term “comprising” or “comprises” is intended to meanthat the compositions and methods include the recited elements, but notexcluding others, “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the stated purpose. Thus,a composition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention.“Consisting of shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

As used herein, the term “about” modifying any amount refers to thevariation in that amount encountered in real world conditions ofproducing sugars and ethanol, e.g., in the lab, pilot plant, orproduction facility. For example, an amount of an ingredient employed ina mixture when modified by “about” includes the variation and degree ofcare typically employed in measuring in an ethanol production plant orlab. For example, the amount of a component of a product when modifiedby “about” includes the variation between batches in an ethanolproduction plant or lab and the variation inherent in the analyticalmethod. Whether or not modified by “about,” the amounts includeequivalents to those amounts. Any quantity stated herein and modified by“about” can also be employed in the present invention as the amount notmodified by “about.” For instance, the term “about” when used before anumerical designation, e.g., temperature, time, amount, andconcentration, including range, indicates approximations which may varyby (+) or (−) 10%, 5% or 1%,

As used herein, the term “unrefined vegetable oil” refers to vegetableoil which has not been subjected to a refining process, such as alkalirefining or physical refining (i.e., distillation, deodorization,bleaching, etc.),

As used herein, the term “free fatty acid” (FFA) refers to anunesterified fatty acid, or more specifically, a fatty acid having acarboxylic acid head and a saturated or unsaturated unbranched aliphatictail (group) of from 4 to 28 carbons. The term “aliphatic” has itgenerally recognized meaning and refers to a group containing onlycarbon and hydrogen atoms which is straight chain, branched chain,cyclic, saturated or unsaturated but not aromatic. In contrast, a fattyacid ester, such as a fatty acid ethyl ester (FAEE), is an esterified(not free) fatty acid. For example, FAEE is a fatty acid esterified withethanol.

As used herein, the term “moisture content” refers to the amount ofwater and other soluble components in the oil composition. The moisturein the vegetable oil composition contains the alkali and/or alkalinemetal, and may contain other soluble components, such as volatilematerial including hexane, ethanol, methanol, and the like.

As used herein, the term “an alkali metal ion” refers to one or moremetal ion of Group 1 of the periodic table (e.g., lithium (L⁺), sodium(Na⁺), potassium (K⁺), etc.).

As used herein, the term “an alkaline metal ion” refers to a metal ionof Group 2 of the periodic table (e.g., magnesium (Mg²⁺), calcium(Ca²⁺), etc.).

As used herein, the term “insoluble” refers to material in the oil whichis not solvated by the aqueous portion, the oil or the moisture contentwithin the oil.

As used herein, the term “unsaponiftables” refers to components of theoil that do not form soaps when blended with a base, and includes anyvariety of possible non-triglyceride materials. This material can act ascontaminants during biodiesel production. Unsaponifiable material cansignificantly reduce the end product yields of the oil composition andcan, in turn, reduce end product yields of the methods disclosed herein.

As used herein, the term “peroxide value” refers to the amount ofperoxide oxygen (in millimoles) per 1 kilogram of fat or oil and is atest of the oxidation of the double bonds of the oils. The peroxidevalue is determined by measuring the amount of iodine (I⁻) viacolorimetry which is formed by the reaction of peroxides (ROOH) formedin the oil with iodide via the following equation: 2

I⁻+H₂O+ROOH->ROH+2OH⁻+I₂.

As used herein, the term “oxidative stability index value” refers to thelength of time the oil resists oxidation at a given temperature.Typically, the oxidation of oil is slow, until the natural resistance(due to the degree of saturation, natural or added antioxidants, etc.)is overcome, at which point oxidation accelerates and becomes veryrapid. The measurement of this time is the oxidative stability indexvalue.

As used herein, the term “vegetable fermentation residue” refers to theresidual components of a vegetable fermentation process after theethanol has been recovered, typically via distillation. Typically, thevegetable fermentation residue comprises water, any residual starch,enzymes, etc.

As used herein, the term “syrup” refers to the viscous composition whichis provided by the evaporation of the thin stillage.

As used herein, the term “base” refers to a compound or compositionwhich raises the pH of an aqueous solution. Suitable bases for use inthis invention include, but are not limited to, sodium hydroxide,potassium hydroxide, calcium hydroxide, or spent alkali wash solution.

As used herein, the term “alkali wash solution” refers to the basicsolution which is used to disinfect the fermentor after the fermentationprocess has been completed. The alkali wash solution typically comprisessodium hydroxide.

As used herein, the phrase “without cooking” refers to a process forconverting starch to ethanol without heat treatment for gelatinizationand dextrinization of starch using alpha-amylase. Generally, for theprocess of the present invention, “without cooking” refers tomaintaining a temperature below starch gelatinization temperatures, sothat saccharification occurs directly from the raw native insolublestarch to soluble glucose while bypassing conventional starchgelatinization conditions. Starch gelatinization temperatures aretypically in a range of 57° C. to 93° C. depending on the starch sourceand polymer type. In the method of the present invention, dextrinizationof starch using conventional liquefaction techniques is not necessaryfor efficient fermentation of the carbohydrate in the grain.

As used herein, the phrase “plant material” refers to all or part of anyplant (e.g., cereal grain), typically a material including starch.Suitable plant material includes grains such as maize (corn, e.g., wholeground corn), sorghum (milo), barley, wheat, rye, rice, and millet; andstarchy root crops, tubers, or roots such as sweet potato and cassava.The plant material can be a mixture of such materials and byproducts ofsuch materials, e.g., corn fiber, corn cobs, stover, or other celluloseand hemicellulose containing materials such as wood or plant residues.Suitable plant materials include corn, either standard corn or waxycorn.

As used herein, the terms “saccharification” and “saccharifying” referto the process of converting starch to smaller polysaccharides andeventually to monosaccharides, such as glucose. Conventionalsaccharification uses liquefaction of gelatinized starch to createsoluble dextrinized substrate which glucoamylase enzyme hydrolyzes toglucose. In the present method, saccharification refers to convertingraw starch to glucose with enzymes, e.g., glucoamylase and acid fungalamylase (AFAU). According to the present method, the raw starch is notsubjected to conventional liquefaction and gelatinization to create aconventional dextrinized substrate.

As used herein, a unit of acid fungal amylase activity (AFAU) refers tothe standard Novozymes units for measuring acid fungal amylase activity.The Novozymes units are described in a Novozymes technical bulletin SOPNo.: EB-SM-0259.02/01. Such units can be measured by detecting productsof starch degradation by iodine titration. 1 unit is defined as theamount of enzyme that degrades 5.260 mg starch dry matter per hour understandard conditions.

As used herein, a unit of glucoamylase activity (GAU) refers to thestandard Novozymes units for measuring glucoamylase activity. TheNovozymes units and assays for determining glucoamylase activity aredescribed in a publicly available Novozymes technical bulletin.

As used herein, a unit of amyloglucosidase activity (AGU) refers to thestandard Novozymes units for measuring amyloglucosidase activity. TheNovozymes units are described in a Novozymes technical bulletin SOP No.:EB-SM-0131.02/01. Such units can be measured by detecting conversion ofmaltose to glucose. The glucose can be determined using the glucosedehydrogenase reaction. 1 unit is defined as the amount of enzyme thatcatalyzes the conversion of 1 mmol maltose per minute under the givenconditions.

Long-chain lipase units (LCLU) refers to the standard Novozymes unitsfor measuring lipase activity. These units are described in patentapplication, WO 2015181308 A1. Such units can be measured by detectingthe hydrolysis product, p-nitrophenol (PNP), of PNP-palmitate andmeasuring its resulting absorbance at 405 nm. 1 unit is defined as theamount of enzyme to release 1 μmol of PNP per minute. However, as usedherein, the amount of lipase dosed in fermentation was based upon thetotal weight of fat within the corn present in fermentation (e.g., 0.4%lipase by weight of corn fat).

Bitumen as used herein can be or include any type of bitumen orbituminous material but does not include aggregates. For example, thebitumen can include bitumen that occurs in nature, bitumen recoveredduring the processing of crude oil and/or other heavy hydrocarbons,and/or bitumen synthetically produced. As used herein, and unlessotherwise specified “asphalt” may refer to a composition having bitumen(no aggregates) or having recycled asphalt (having aggregates), or acombination thereof, e.g., a combination of virgin asphalt (bitumen) andrecycled asphalt.

Exemplary Method for Converting Starch to Ethanol

The present disclosure provides methods for producing high levels oflower alkyl esters in vegetable oil, e.g., during fermentation of plantmaterial, and to the vegetable oil composition produced thereby. Thelower alkyl ester content may be the result of fermentation, e.g., asimultaneous fermentation/transesterification, which may be enhanced byadding an esterase, or may be the result of adding lower alkyl esters toa vegetable oil composition or using an enzyme catalyzed or chemicalacid/base catalyzed transesterification reaction, to increase loweralkyl ester content. The present disclosure also relates to methods forusing the vegetable oil compositions.

The method converts starch from plant material to ethanol. In anembodiment, the present method can include preparing the plant materialfor saccharification, converting the prepared plant material to sugarswithout cooking, and fermenting the sugars.

The plant material can be prepared for saccharification by any a varietyof methods, e.g., by grinding, to make the starch available forsaccharification and fermentation. In an embodiment, the vegetablematerial can be ground so that a substantial portion, e.g., a majority,of the ground material fits through a sieve with a 0.1-0.5 mm screen.For example, in an embodiment, about 70% or more, of the groundvegetable material can fit through a sieve with a 0.1-0.5 mm screen. Inan embodiment, the reduced plant material can be mixed with liquid atabout 20 to about 50 wt-% or about 25 to about 45 wt-% dry reduced plantmaterial.

The process can include converting reduced plant material to sugars thatcan be fermented by a microorganism such as yeast. This conversion canbe effected by saccharifying the reduced plant material with an enzymepreparation, such as a saccharifying enzyme composition. A saccharifyingenzyme composition can include any of a variety of known enzymessuitable for converting reduced plant material to fermentable sugars,such as amylases (e.g., α-amylase and/or glucoamylase). In anembodiment, saccharification is conducted at a pH of about 6.0 or less,for example, about 4.5 to about 5.0.

The process includes fermenting sugars from reduced plant material toethanol. Fermenting can be effected by a microorganism, such as yeast.In an embodiment, fermentation is conducted at a pH of about 6 or less,for example, about 4.5 to about 5. In an embodiment, the present methodcan include varying the pH. For example, fermentation can includefilling the fermenter at pH of about 3 to about 4.5 during the firsthalf of fill and at a pH of about 4.5 to about 6 during the second halfof the fermenter fill cycle. In an embodiment, fermentation is conductedat a temperature of about 25 to about 40° C. or about 30 to about 35° C.In an embodiment, during fermentation the temperature is decreased fromabout 40° C. to about 30° C. or about 25° C., or from about 35° C. toabout 30° C., during the first half of the fermentation, and thetemperature is held at the lower temperature for the second half of thefermentation. In an embodiment, fermentation is conducted for about to25 (e.g., 24) to about to 150 hours, for example, for about 48 (e.g.,47) to about 96 hours.

The process can include simultaneously converting reduced plant materialto sugars and fermenting those sugars with a microorganism such asyeast.

The product of the fermentation process is referred to herein as “beer.”Ethanol can be recovered from the fermentation mixture, from the beer,by any of a variety of known processes, such as by distilling. Theremaining stillage includes both liquid and solid material. The liquidand solid can be separated by, for example, centrifugation.

Preparing the Plant Material

The method converts starch from plant material to ethanol and vegetableoil. The plant material can be reduced by a variety of methods, e.g., bygrinding, to make the starch available for saccharification andfermentation. Other methods of plant material reduction are available.For example, vegetable material, such as kernels of corn, can be groundwith a ball mill, a roller mill, a hammer mill, or another mill knownfor grinding vegetable material, and/or other materials for the purposesof particle size reduction. The use of emulsion technology, rotarypulsation, and other means of particle size reduction can be employed toincrease surface area of plant material while raising the effectivenessof flowing the liquefied media. The prepared plant material can bereferred to as being or including “raw starch.”

A fine grind exposes more surface area of the plant material, orvegetable material, and can facilitate saccharification andfermentation. In an embodiment, the vegetable material is ground so thata substantial portion, e.g., a majority, of the ground material fitsthrough a sieve with a 0.1-0.5 mm screen. In an embodiment, about 35% ormore of the ground vegetable material can fit through a sieve with a0.1-0.5 mm screen. In an embodiment, about 35 to about 70% of the groundvegetable material can fit through a sieve with a 0.1-0.5 mm screen. Inan embodiment, about 50% or more of the ground vegetable material canfit through a sieve with a 0.1-0.5 mm screen. In an embodiment, about90% of the ground vegetable material can fit through a sieve with a0.1-0.5 mm screen. In an embodiment, all of the ground vegetablematerial can fit through a sieve with a 0.1-0.5 mm screen.

Fractionation

In an embodiment, the vegetable material can be fractionated into one ormore components. For example, a vegetable material such as a cerealgrain or corn can be fractionated into components such as fiber (e.g.,corn fiber), germ (e.g., corn germ), and a mixture of starch and protein(e.g., a mixture of corn starch and corn protein). One or a mixture ofthese components can be fermented as described herein. Fractionation ofcorn or another plant material can be accomplished by any of a varietyof methods or apparatus. For example, a system manufactured by Satakecan be used to fractionate plant material such as corn.

Saccharification

The process can include converting reduced plant material to sugars thatcan be fermented by a microorganism such as yeast. This conversion canbe effected by saccharifying the reduced plant material with any of avariety of known saccharifying enzyme compositions. In an embodiment,the saccharifying enzyme composition includes an amylase, such as analpha amylase (e.g., acid fungal amylase). The enzyme preparation canalso include glucoamylase. The enzyme preparation need not, and, in anembodiment, does not include protease. However, ethanol productionmethods can conserve water by reusing process waters (backset) which maycontain protease. In an embodiment, the method employs acid fungalamylase for hydrolyzing raw starch.

Saccharifying can be conducted without cooking. For example,saccharifying can be conducted by mixing source of saccharifying enzymecomposition (e.g., commercial enzyme), yeast, and fermentationingredients with ground grain and process waters without cooking.

In an embodiment, saccharifying can include mixing the reduced plantmaterial with a liquid, which can form a slurry or suspension and addingsaccharifying enzyme composition (e.g., at least one of acid fungalamylase and glucoamylase) to the liquid. In an embodiment, the methodincludes mixing the reduced plant material and liquid and then addingthe saccharifying enzyme composition (e.g., at least one of acid fungalamylase and glucoamylase). Alternatively, adding enzyme composition canprecede or occur simultaneously with mixing.

In an embodiment, the reduced plant material can be mixed with liquid atabout 20 to about 50 wt-%, about 25 to about 45 (e.g., 44) wt-%, about30 to about 40 (e.g., 39) wt-%, or about 35 wt-% dry reduced plantmaterial. As used herein, wt-% of reduced plant material in a liquidrefers to the percentage of dry substance reduced plant material or drysolids. In an embodiment, the method can convert raw or native starch(e.g., in dry reduced plant material) to ethanol at a faster rate athigher dry solids levels compared to conventional saccharification withcooking. The method may be practiced at higher dry solids levelsbecause, unlike the conventional process, it does not includegelatinization, which increases viscosity.

Suitable liquids include water and a mixture of water and processwaters, such as stillage (backset), scrubber water, evaporatorcondensate or distillate, side stripper water from distillation, orother ethanol plant process waters. In an embodiment, the liquidincludes water. In an embodiment, the liquid includes water in a mixturewith about 1 to about 70 vol-% stillage, about 15 to about 60 vol-%stillage, about 30 to about 50 vol-% stillage, or about 40 vol-%stillage.

In an embodiment, the method employs a preparation of plant materialthat supplies sufficient quantity and quality of nitrogen for efficientfermentation under high gravity conditions (e.g., in the presence ofhigh levels of reduced plant material). Thus, in an embodiment, no oronly low levels of stillage can suffice.

The method may produce lower viscosity stillage. Therefore, in anembodiment, increased levels of stillage can be employed withoutdetrimental increases in viscosity of the fermentation mixture orresulting stillage.

The present process may avoid temperature induced Maillard Reactions andprovides increased levels of FAN in the reduced plant material, whichare effectively utilized by the yeast in fermentation.

Saccharification can employ any of a variety of known enzyme sources(e.g., a microorganism) or compositions to produce fermentable sugarsfrom the reduced plant material. In an embodiment, the saccharifyingenzyme composition includes an amylase, such as an alpha amylase (e.g.,acid fungal amylase) or a glucoamylase.

In certain embodiments, the method employs an esterase defined by EC3.1.1.1 (a carboxylic-ester hydrolase) or 3.1.1.3 (a triacylglycerollipase).

In certain embodiments, saccharification is conducted without pHadjustment.

In an embodiment, saccharification is conducted at a pH of about 6.0 orless, pH of about 3.0 to about 6.0, about 3.5 to about 6.0, about 4.0 toabout 5.0, about 4.0 to about 4.5, or about 4.5 to about 5.0. Theinitial pH of the saccharification mixture can be adjusted by additionof, for example, ammonia, sulfuric acid, phosphoric acid, process waters(e.g., stillage (backset), evaporator condensate (distillate), sidestripper bottoms, and the like), and the like. Activity of certainsaccharifying enzyme compositions (e.g., at least one of acid fungalamylase and glucoamylase) can be enhanced at pH lower than the aboveranges.

In an embodiment, saccharification is conducted at a temperature ofabout 25 to about 40° C. or about 30 to about 35° C.

In an embodiment, saccharifying can be carried out employing quantitiesof saccharifying enzyme composition (e.g., at least one of acid fungalamylase and glucoamylase) selected to maintain low concentrations ofdextrin in the fermentation broth. For example, the process can employquantities of saccharifying enzyme composition (e.g., at least one ofacid fungal amylase and glucoamylase) selected to maintain maltotriose(DP3) at levels at or below about 0.2 wt-% or at or below about 0.1wt-%. For example, the present process can employ quantities ofsaccharifying enzyme composition (e.g., at least one of acid fungalamylase and glucoamylase) selected to maintain dextrin with a degree ofpolymerization of 4 or more (DP4+) at levels at or below about 1 wt-% orat or below about 0.5 wt %. For maintaining low levels of maltotrioseand/or DP4+, suitable levels of acid fungal amylase and glucoamylaseinclude about 0.3 to about 3 AFAU/gram dry solids reduced plant material(e.g., DSC) of acid fungal amylase and about 1 to about 2.5 (e.g., 2.4)AGU per gram dry solids reduced plant material (e.g., DSC) ofglucoamylase. In an embodiment, the reaction mixture includes about 1 toabout 2 MAU/gram dry solids reduced plant material (e.g., DSC) of acidfungal amylase and about 1 to about 1.5 AGU per gram dry solids reducedplant material (e.g., DSC) of glucoamylase.

In an embodiment, saccharifying can be carried out employing quantitiesof saccharifying enzyme composition (e.g., at least one of acid fungalamylase and glucoamylase) selected to maintain low concentrations ofmaltose in the fermentation broth. For example, the present process canemploy quantities of saccharifying enzyme composition (e.g., at leastone of acid fungal amylase and glucoamylase) selected to maintainmaltose at levels at or below about 0.3 wt %. For maintaining low levelsof maltose, suitable levels of acid fungal amylase and glucoamylaseinclude about 0.3 to about 3 AFAU/gram dry solids reduced plant material(e.g., DSC) of acid fungal amylase and about 1 to about 2.5 (e.g., 2.4)AGU per gram dry solids reduced plant material (e.g., DSC) ofglucoamylase. In an embodiment, the reaction mixture includes about 1 toabout 2 MAU/gram dry solids reduced plant material (e.g., DSC) of acidfungal amylase and about 1 to about 1.5 AGU per gram dry solids reducedplant material (e.g., DSC) of glucoamylase.

Acid Fundal Amylase

In certain embodiments, the method employs an a-amylase. The x-amylasecan be one produced by fungi. The α-amylase can be one characterized byits ability to hydrolyze carbohydrates under acidic conditions. Anamylase produced by fungi and able to hydrolyze carbohydrates underacidic conditions is referred to herein as acid fungal amylase, and isalso known as an acid stable fungal α-amylase. Acid fungal amylase cancatalyze the hydrolysis of partially hydrolyzed starch and largeoligosaccharides to sugars such as glucose. The acid fungal amylase thatcan be employed in the process can be characterized by its ability toaid the hydrolysis of raw or native starch, enhancing thesaccharification provided by glucoamylase. In an embodiment, the acidfungal amylase produces more maltose than conventional (e.g., bacterial)alpha-amylases.

Suitable acid fungal amylase can be isolated from any of a variety offungal species, including Aspergiilus, Rhizopus, Mucor, Candida,Coriolus, Endothia, Enthomophtora, Irpex, Penicillium, Sclerotium andTorulopsis species. In an embodiment, the acid fungal amylase isthermally stable and is isolated from Aspergillus species, such as A.niger, A. saitoi or A. oryzae, from Mucor species such as M. pusillus orM. miehei, or from Endothia species such as E. parasitica. In anembodiment, the acid fungal amylase is isolated from Aspergillus niger.The acid fungal amylase activity can be supplied as an activity in aglucoamylase preparation, or it can be added as a separate enzyme. Asuitable acid fungal amylase can be obtained from Novozymes, for examplein combination with glucoamylase.

The amount of acid fungal amylase employed in the present process canvary according to the enzymatic activity of the amylase preparation.Suitable amounts include about 0.1 to about 10 acid fungal amylase units(AFAU) per gram of dry solids reduced plant material (e.g., dry solidscorn (DSC)). In an embodiment, the reaction mixture can include about0.3 to about 3 AFAU/gram dry solids reduced plant material (e.g., DSC).In an embodiment, the reaction mixture can include about 1 to about 2AFAU/gram dry solids reduced plant material (e.g., DSC).

Glucoamylase

In certain embodiments, the method can employ a glucoamylase.Glucoamylase is also known as amyloglucosidase and has the systematicname 1,4-alpha-D-glucan glucohydrolase (E.C. 3.2.1.3). Glucoamylaserefers to an enzyme that removes successive glucose units from thenon-reducing ends of starch. For example, certain glucoamylases canhydrolyze both the linear and branched glucosidic linkages of starch,amylose, and amylopectin. A variety of suitable glucoamylases are knownand commercially available. For example, suppliers such as Novozymes andGenencor provide glucoamylases. The glucoamylase can be of fungalorigin.

The amount of glucoamylase employed in the present process can varyaccording to the enzymatic activity of the amylase preparation. Suitableamounts include about 0.1 to about 6.0 glucoamylase units (AGU) per gramdry solids reduced plant material (e.g., DSC). In an embodiment, thereaction mixture can include about 1 to about 3 AGU per gram dry solidsreduced plant material (e.g., DSC). In an embodiment, the reactionmixture can include about 1 to about 2.5 (e.g., 2.4) AGU per gram drysolids reduced plant material (e.g., DSC). In an embodiment, thereaction mixture can include about 1 to about 2 AGU per gram dry solidsreduced plant material (e.g., DSC). In an embodiment, the reactionmixture can include about 1 to about 1.5 AGU per gram dry solids reducedplant material (e.g., DSC). In an embodiment, the reaction mixture caninclude about 1.2 to about 1.5 AGU per gram dry solids reduced plantmaterial (e.g., DSC).

Fermentation

The process includes fermenting sugars from reduced plant material toethanol. Fermenting can be effected by a microorganism, such as yeast.The fermentation mixture need not, and in an embodiment does not,include protease. However, the process waters may contain protease. Theamount of protease can be less than that used in the conventionalprocess. In one embodiment, fermenting is conducted on a starchcomposition that has not been cooked. In an embodiment, the fermentationprocess produces potable alcohol. Potable alcohol has only acceptable,nontoxic levels of other alcohols, such as fusel oils. Fermenting caninclude contacting a mixture including sugars from the reduced plantmaterial with yeast under conditions suitable for growth of the yeastand production of ethanol. In an embodiment, fermenting employs thesaccharification mixture.

Any of a variety of yeasts can be employed as the yeast starter in thepresent process. Suitable yeasts include any of a variety ofcommercially available yeasts, such as commercial strains ofSaccharomyces cerevisiae. Suitable strains include “Fali”(Fleischmann's), Thermosac (Alltech), Ethanol Red (LeSafre), BioFerm AFT(North American Bioproducts), and the like. In an embodiment, the yeastis selected to provide rapid growth and fermentation rates in thepresence of high temperature and high ethanol levels. In an embodiment,Fali yeast has been found to provide good performance as measured byfinal alcohol content of greater than 17% by volume.

The amount of yeast starter employed is selected to effectively producea commercially significant quantity of ethanol in a suitable time, e.g.,less than 75 hours.

Yeast can be added to the fermentation by any of a variety of methodsknown for adding yeast to fermentation processes. For example, yeaststarter can be added by as a dry batch, or by conditioning/propagating.In an embodiment, yeast starter is added as a single inoculation. In anembodiment, yeast is added to the fermentation during the fermenter fillat a rate of 5 to 100 pounds of active dry yeast (ADY) per 100,000gallons of fermentation mash. In an embodiment, the yeast can beacclimated or conditioned by incubating about 5 to 50 pounds of ADY per10,000 gallon volume of fermenter volume in a prefermenter orpropagation tank. Incubation can be from 8 to 16 hours during thepropagation stage, which is also aerated to encourage yeast growth. Theprefermenter used to inoculate the main fermenter is can be from 1 to10% by volume capacity of the main fermenter, for example, from 2.5 to5% by volume capacity relative to the main fermenter.

In an embodiment, the fermentation is conducted at a pH of about 6 orless, pH of about 3 to about 6, about 3.5 to about 6, about 4 to about5, about 4 to about 4.5, or about 4.5 to about 5. The initial pH of thefermentation mixture can be adjusted by addition of, for example,ammonia, sulfuric acid, phosphoric acid, process waters (e.g., stillage(backset), evaporator condensate (distillate), side stripper bottoms,and the like), and the like.

Distillery yeast grow well over the pH range of 3 to 6, but are moretolerant of lower pH's down to 3.0 than most contaminant bacterialstrains. Contaminating lactic and acetic acid bacteria grow best at pHof 5.0 and above. Thus, in the pH range of 3.0 to 3.5, it is believedthat ethanol fermentation will predominate because yeast will growbetter than contaminating bacteria.

In an embodiment, the method can include varying the pH. It is believedthat varying the pH can be conducted to reduce the likelihood ofcontamination early in fermentation and/or to increase yeast growth andfermentation during the latter stages of fermentation. For example,fermentation can include filling the fermenter at pH of about 3 to about4.5 during the first half of fill. Fermentation can include increasingthe slurry pH to pH of about 4.5 to about 6 during the second half ofthe fermenter fill cycle. Fermentation can include maintaining pH byadding fresh substrate slurry at the desired pH as described above. Inan embodiment, during fermentation (after filling), pH is not adjusted.Rather, in this embodiment, the pH is determined by the pH of thecomponents during filling.

In an embodiment, the pH is decreased to about five (5) or below in thecorn process waters. In an embodiment, the pH is about pH 4 (e.g., 4.1)at the start of fermentation fill and is increased to about pH 5 (e.g.,5.2) toward the end of fermentation fill. In an embodiment, the methodincludes stopping pH control of the mash slurry after the yeast culturebecomes established during the initial process of filling the fermenter,and then allowing the pH to drift up in the corn process waters duringthe end stages of filling the fermenter.

In an embodiment, fermentation is conducted for about to 25 (e.g., 24)to about to 150 hours, about 25 (e.g., 24) to about 96 hours, about 40to about 96 hours, about 45 (e.g., 44) to about 96 hours, about 48(e.g., 47) to about 96 hours. For example, fermentation can be conductedfor about 30, about 40, about 50, about 60, or about 70 hours. Forexample, fermentation can be conducted for about 35, about 45, about 55,about 65, or about 75 hours.

In an embodiment, fermentation is conducted at a temperature of about 25to about 40° C. or about 30 to about 35° C., In an embodiment, duringfermentation the temperature is decreased from about 40° C. to about 30°C. or about 25° C., or from about 35° C. to about 30° C., during thefirst half of the fermentation, and the temperature is held at the lowertemperature for the second half of the fermentation. In an embodiment,the temperature can be decreased as ethanol is produced. For example, inan embodiment, during fermentation the temperature can be as high asabout 99° F. and then reduced to about 79° F. This temperature reductioncan be coordinated with increased ethanol titers (%) in the fermenter.

In an embodiment, the method includes solids staging. Solids stagingincludes filling at a disproportionately higher level of solids duringthe initial phase of the fermenter fill cycle to increase initialfermentation rates. The solids concentration of the mash entering thefermenter can then be decreased as ethanol titers increase and/or as thefermenter fill cycle nears completion. In an embodiment, the solidsconcentration can be about 40% (e.g., 41%) during the first half of thefermentation fill. This can be decreased to about 25% after thefermenter is 50% lull and continuing until the fermenter fill cycle isconcluded. In the above example, such a strategy results in a fullfermenter with solids at 33%. It is believed that solids staging canaccelerate enzyme hydrolysis rates and encourage a rapid onset tofermentation by using higher initial fill solids. It is believed thatlowering solids in the last half of fill can reduce osmotic pressurerelated stress effects on the yeast. By maintaining overall fermenterfill solids within a specified range of fermentability, solids stagingimproves the capacity of the yeast to ferment high gravity mashes towardthe end of fermentation.

Esterase

In certain embodiments, the method employs an esterase assigned to IUBEC 3.1.1.1 or EC 3.1.1.3. In certain embodiments, the method employs anesterase such as a lipase.

Exemplary esterases include but are not limited to lipases such as thosefrom plant, fungi, yeast or bacteria, e.g., lipases from filamentousfungi, such as those of genera Rhizopus, Mucor, Geotrichum, Aspergillus,Fusarium and Penicillium, as well as bacteria such as Bacilluscoagulans, Bacillus stearothermophilus, Bacillus alcalophilusPseudomonas sp., Pseudomonas aeruginosa, Burkholderia multivorans,Burkholderia cepacia, Staphylococcus caseolyticus, and yeast such asCandida rugosa. Candida tropicalis, Candida antarctica, Candidacylindracea, Candida parapsilopsis, Candida deformans, Candida curvata,Candida valida, Yarrowia lipolytica, Rhodotorula giutinis. Rhodotoruiapilimomae, Pichia bispora, Pichia mexicana, Pichia sivicola,Saccharomyces cerevisiae, Candida wickerhamii, Williopsis californica,and Candida boidinii. The amount of esterase may be from about 0.01% toabout 20% w/w of vegetable fat, e.g., from about 0.02% to about 0.2% w/wof vegetable fat, about 0.04% to about 4% w/w of vegetable fat, about 2%to about 20% w/w of vegetable fat, or about 0.03% to about 0.5% w/w ofvegetable fat.

In one embodiment, the esterase is added when fermentation is initiated,after fermentation is initiated, when fermentation is complete, or anycombination thereof. In one embodiment, the esterase is in an amountthat is at least 0.01% w/w of the weight of plant fat in the aqueouscomposition prior to fermentation In one embodiment, the esterase is inan amount that is at least 0.4% w/w of the weight of plant fat in theaqueous composition prior to fermentation. In one embodiment, theesterase is in an amount that is at least 1% w/w of the weight of plantfat in the aqueous composition prior to fermentation.

In one embodiment, the esterase is in an amount that is at least 4% w/wof the weight of plant fat in the aqueous composition prior tofermentation. In one embodiment, the saccharification preceding thefermentation is not pH adjusted,

Simultaneous Saccharification and Fermentation

The process can include simultaneously converting reduced plant materialto sugars and fermenting those sugars with a microorganism such asyeast. Simultaneous saccharifying and fermenting can be conducted usingthe reagents and conditions described above for saccharifying andfermenting.

In an embodiment, saccharification and fermentation is conducted at atemperature of about 25 to about 40° C. or about 30 to about 35° C. Inan embodiment, during saccharification and fermentation the temperatureis decreased from about 40 to about 25° C. or from about 35 to about 30°C. during the first half of the saccharification, and the temperature isheld at the lower temperature for the second half of thesaccharification.

Higher temperatures early during saccharification and fermentation mayincrease conversion of starch to fermentable sugar when ethanolconcentrations are low. This can aid in increasing ethanol yield. Athigher ethanol concentrations, this alcohol can adversely affect theyeast. Thus, it is believed that lower temperatures later duringsaccharification and fermentation are beneficial to decrease stress onthe yeast. This can aid in increasing ethanol yield.

Higher temperatures early during saccharification and fermentation mayreduce viscosity during at least a portion of the fermentation. This canaid in temperature control. Lower temperatures later duringsaccharification and fermentation may be beneficial to reduce theformation of glucose after the yeast has stopped fermenting. Glucoseformation late in fermentation can be detrimental to the color of thedistillers dried grain co-product.

In an embodiment, saccharification and fermentation is conducted at a pHof about 6 or less, pH of about 3 to about 6, about 3.5 to about 6,about 4 to about 5, about 4 to about 4.5, or about 4.5 to about 5. Theinitial pH of the saccharification and fermentation mixture can beadjusted by addition of, for example, ammonia, sulfuric acid, phosphoricacid, process waters (e.g., stillage (backset), evaporator condensate(distillate), side stripper bottoms, and the like), and the like.

In an embodiment, saccharification and fermentation is conducted forabout to 25 (e.g., 24) to about to 150 hours, about 25 (e.g., 24) toabout 72 hours, about 45 to about 55 hours, about 50 (e.g., 48) to about96 hours, about 50 to about 75 hours, or about 60 to about 70 hours. Forexample, saccharification and fermentation can be conducted for about30, about 40, about 50, about 60, or about 70 hours. For example,saccharification and fermentation can be conducted for about 35, about45, about 55, about 65, or about 75 hours.

In an embodiment, simultaneous saccharifying and fermenting can becarried out employing quantities of enzyme and yeast selected tomaintain high concentrations of yeast and high levels of budding of theyeast in the fermentation broth. For example, the present process canemploy quantities of enzyme and yeast selected to maintain yeast at orabove about 300 cells/mL or at about 300 to about 600 cells/mL.

In an embodiment, simultaneous saccharifying and fermenting can becarried out employing quantities of enzyme and yeast selected foreffective fermentation without added exogenous nitrogen; without addedprotease; and/or without added backset. Backset can be added, ifdesired, to consume process water and reduce the amount of wastewaterproduced by the process. In addition, the present process maintains lowviscosity during saccharifying and fermenting.

For example, simultaneous saccharifying and fermenting can employ acidfungal amylase at about 0.1 to about 10 AFAU per gram of dry solidsreduced plant material (e.g., DSC), glucoamylase at about 0.5 to about 6AGU per gram dry solids reduced plant material (e.g., DSC) and anesterase as described herein. For example, simultaneous saccharifyingand fermenting can employ acid fungal amylase at about 0.3 to about 3AFAU per gram of dry solids reduced plant material (e.g., DSC),glucoamylase at about 1 to about 3 AGU per gram dry solids reduced plantmaterial (e.g., DSC) and an esterase as described herein. For example,simultaneous saccharifying and fermenting can employ acid fungal amylaseat about 1 to about 2 AFAU per gram of dry solids reduced plant material(e.g., DSC), glucoamylase at about 1 to about 1.5 AGU per gram drysolids reduced plant material (e.g., DSC) and an esterase as describedherein.

In an embodiment, simultaneous saccharifying and fermenting can becarried out employing quantities of enzyme and yeast selected tomaintain low concentrations of glucose in the fermentation broth. Forexample, the present process can employ quantities of enzyme and yeastselected to maintain glucose at levels at or below about 2 wt-%, at orbelow about 1 wt-%, at or below about 0.5 wt-%, or at or below about 0.1wt-%. For example, the present process can employ quantities of enzymeand yeast selected to maintain glucose at levels at or below about 2wt-% during saccharifying and fermenting. For example, the presentprocess can employ quantities of enzyme and yeast selected to maintainglucose al levels at or below about 2 wt-% from hours 0-10 (or from 0 toabout 15% of the time) of saccharifying and fermenting. For example, thepresent process can employ quantities of enzyme and yeast selected tomaintain glucose at levels at or below about 1 wt-%, at or below about0.5 wt-%, or at or below about 0.1 wt-% from hours 12-54 (or from about15% to about 80% of the time) of saccharifying and fermenting. Forexample, the present process can employ quantities of enzyme and yeastselected to maintain glucose at levels at or below about 1 wt-% fromhours 54-66 (or about from 80% to about 100% of the time) ofsaccharifying and fermenting. Suitable levels of enzyme include acidfungal amylase at about 0.3 to about 3 AFAU per gram of dry solidsreduced plant material (e.g., DSC) and glucoamylase at about 1 to about3 AGU per gram dry solids reduced plant material (e.g., DSC). Forexample, simultaneous saccharifying and fermenting can employ acidfungal amylase at about 1 to about 2 AFAU per gram of dry solids reducedplant material (e.g., DSC), glucoamylase at about 1 to about 1.5 AGU pergram dry solids reduced plant material (e.g., DSC) and an esterase asdescribed herein.

In an embodiment, simultaneous saccharifying and fermenting can becarried out employing quantities of enzyme and yeast selected tomaintain low concentrations of maltose (DP2) in the fermentation broth.For example, the present process can employ quantities of enzyme andyeast selected to maintain maltose at levels at or below about 0.5 wt-%or at or below about 0.2 wt-%. Suitable levels of enzyme include acidfungal amylase at about 0.3 to about 3 AFAU per gram of dry solidsreduced plant material (e.g., DSC), glucoamylase at about 1 to about 3AGU per gram dry solids reduced plant material (e.g., DSC) and anesterase as described herein. For example, simultaneous saccharifyingand fermenting can employ acid fungal amylase at about 1 to about 2 AFAUper gram of dry solids reduced plant material (e.g., DSC), glucoamylaseat about 1 to about 1.5 AGU per gram dry solids reduced plant material(e.g., DSC) and an esterase as described herein.

In an embodiment, simultaneous saccharifying and fermenting can becarried out employing quantities of enzyme and yeast selected tomaintain low concentrations of dextrin in the fermentation broth. Forexample, the present process can employ quantities of enzyme and yeastselected to maintain maltotriose (DP3) at levels at or below about 0.5wt-%, at or below about 0.2 wt-%, or at or below about 0.1 wt-%. Forexample, the present process can employ quantities of enzyme and yeastselected to maintain dextrin with a degree of polymerization of 4 ormore (DP4+) at levels at or below about 1 wt-% or at or below about 0.5wt-%. Suitable levels of enzyme include acid fungal amylase at about 0.3to about 3 AFAU per gram of dry solids reduced plant material (e.g.,DSC), glucoamylase at about 1 to about 3 AGU per gram dry solids reducedplant material (e.g., DSC) and an esterase as described herein. Forexample, simultaneous saccharifying and fermenting can employ acidfungal amylase at about 1 to about 2 AFAU per gram of dry solids reducedplant material (e.g., DSC), glucoamylase at about 1 to about 1.5 AGU pergram dry solids reduced plant material (e.g., DSC) and an esterase asdescribed herein.

In an embodiment, simultaneous saccharifying and fermenting can becarried out employing quantities of enzyme and yeast selected tomaintain low concentrations of fusel oils in the fermentation broth. Forexample, the present process can employ quantities of enzyme and yeastselected to maintain fusel oils at levels at or below about 0.4 to about0.5 wt-%. Suitable levels of enzyme include acid fungal amylase at about0.3 to about 3 AFAU per gram of dry solids reduced plant material (e.g.,DSC), glucoamylase at about 1 to about 3 AGU per gram dry solids reducedplant material (e.g., DSC) and an esterase as described herein. Forexample, simultaneous saccharifying and fermenting can employ acidfungal amylase at about 1 to about 2 AFAU per gram of dry solids reducedplant material (e.g., DSC), glucoamylase at about 1 to about 1.5 AGU pergram dry solids reduced plant material (e.g., DSC) and an esterase asdescribed herein.

Additional Ingredients for Saccharification and/or Fermentation

The saccharification and/or fermentation mixture can include additionalingredients to increase the effectiveness of the process. For example,the mixture can include added nutrients (e.g., yeast micronutrients),antibiotics, salts, added enzymes, and the like. Nutrients can bederived from stillage or backset added to the liquid. Suitable salts caninclude zinc or magnesium salts, such as zinc sulfate, magnesiumsulfate, and the like. Suitable added enzymes include those added toconventional processes, such as protease, phytase, cellulase,hemicellulase, exo- and endo-glucanase, xylanase, and the like.

Recovering Ethanol from the Beer

The product of the fermentation process is referred to herein as “beer”.For example, fermenting corn produces “corn beer”. Ethanol can berecovered from the fermentation mixture, from the beer, by any of avariety of known processes. For example, ethanol can be recovered bydistillation.

The remaining stillage includes both liquid and solid material. Theliquid and solid can be separated by, for example, centrifugation. Therecovered liquid, thin stillage, can be employed as at least part of theliquid for forming the saccharification and fermentation mixture forsubsequent batches or runs.

The recovered solids, distiller's dried grain, include unfermented grainsolids and spent yeast solids. Thin stillage can be concentrated to asyrup, which can be added to the distiller's dried grain and the mixturethen dried to form distiller's dried grain plus solubles. Distiller'sdried grain and/or distiller's dried grain plus solubles can be sold asanimal feed.

Burn-Out of Residual Starches for Subsequent Fermentation

In an embodiment, the present method can include heat treatment of thebeer or stillage, e.g., between the beer well and distillation. Thisheat treatment can convert starches to dextrins and sugars forsubsequent fermentation in a process known as burn-out. Such a treatmentstep can also reduce fouling of distillation trays and evaporator heatexchange surfaces. In an embodiment, heat treatment staging can beperformed on whole stillage. Following enzymatic treatment of theresidual starches, in an embodiment, the resulting dextrins and sugarscan be fermented within the main fermentation process as recycledbackset or processed in a separate fermentation train to produceethanol.

Fractionation of Solids from Fermentation

Large pieces of germ and fiber can ferment the residual starch in thefermenter. After fermentation, the fractions could be removed prior toor after distillation. Removal can be effected with a surface skimmerbefore to distillation. In an embodiment, screening can be performed onthe beer. The screened material can then be separated from theethanol/water mix by, for example, centrifugation and rotary steam drumdrying, which can remove the residual ethanol from the cake. Inembodiments in which the larger fiber and germ pieces are removed priorto bulk beer distillation, a separate stripper column for the Fiber/germstream can be utilized. Alternatively, fiber and germ could be removedby screening the whole stillage after distillation.

In an embodiment, all the components are blended and dried together. Thefiber and germ can be removed from the finished product by aspirationand/or size classification. The fiber from the DDGS can be aspirated.Removal of fiber by aspiration after drying increased the amount of oiland protein in the residual DOGS by 0.2 to 1.9% and 0.4 to 1.4%,respectively. The amount of NDF in the residual DOGS decreased by 0.1 to2.8%.

In an embodiment, fractionation can employ the larger fiber and germpieces to increase the particle size of that part of the DDGS derivedfrom the endosperm, as well as to improve syrup carrying capacity. Aring dryer disintegrator can provide some particle size reduction andhomogenization.

Continuous Fermentation

The process can be run via a batch or continuous process. A continuousprocess includes moving (pumping) the saccharifying and/or fermentingmixtures through a series of vessels (e.g., tanks) to provide asufficient duration for the process. For example, a multiple stagefermentation system can be employed for a continuous process with 48-96hours residence time. For example, reduced plant material can be fedinto the top of a first vessel for saccharifying and fermenting.Partially incubated and fermented mixture can then be drawn out of thebottom of the first vessel and fed in to the top of a second vessel, andso on.

The method achieves efficient production of high concentrations ofethanol without a liquefaction or saccharification stage prior tofermentation. The method can provide low concentrations of glucose andefficient fermentation. In the present method, it appears that theglucose is consumed rapidly by the fermenting yeast cell. It is believedthat such low glucose levels reduce stress on the yeast, such as stresscaused by osmotic inhibition and bacterial contamination pressures.Ethanol levels greater than 18% by volume can be achieved in about 45 toabout 96 hours,

Exemplary Vegetable Oil Compositions

The oil compositions contain certain levels of lower alkyl esters,making them valuable for use in applications including but not limitedto asphalt rejuvenation, bio-diesel, edible and nutraceuticalapplications. The oil compositions may be recovered from a fermentationprocess, e.g., one that included an added (exogenous) esterase, and maycontain elevated levels of lower alkyl esters, or lower alkyl esters maybe added to the oil compositions or enzyme-catalyzed or chemicalacid/base catalyzed transesterification reactions may be employed toincrease the level of lower alkyl esters in the oil compositions.

In one embodiment, a vegetable oil such as corn oil, soybean oil,sorghum oil or wheat oil is provided by the fermentation of corn,soybean, sorghum or wheat in the production of ethanol. Referring toFIG. 1, in a typical exemplary ethanol production process, corn, forinstance, can be prepared for further treatment in a preparation system.The preparation system may comprise a cleaning or screening step toremove foreign material, such as rocks, dirt, sand, pieces of corn cobsand stalk, and other unfermentable material. After cleaning/screening,the particle size of corn can be reduced by milling to facilitatefurther processing. The corn kernels may also be fractionated intostarch-containing endosperm and fiber and germ. The milled corn orendosperm is then slurried with water, enzymes and agents to facilitatethe conversion of starch into sugar (e.g., glucose). The sugar can thenbe converted into ethanol by an ethanologen (e.g., yeast) in afermentation system. In one embodiment, the fermentation is carried outwithout creating a hot slurry (i.e., without cooking). In such anembodiment, the fermentation includes the step of saccharifying thestarch composition with an enzyme composition to form a saccharifiedcomposition (e.g., without cooking). In one embodiment, the starchcomposition comprises water and from 5% to 60% dried solids granularstarch, based on the total weight of the starch composition. In anotherembodiment, the starch composition comprises 10% to 50% dried solidsgranular starch, or 15% to 40% dried solids granular starch, or 20% to25% dried solids granular starch, based on the total weight of thestarch composition.

The fermentation product is beer, which comprises ethanol, water, oil,additional soluble components, unfermented particulate matter, etc. Thefermentation product can then be distilled to provide ethanol, leavingthe remaining components as whole stillage. The whole stillage can thenbe separated to provide a liquid component (i.e., thin stillage) and asolid component. The solid component can be dried to provide thedistillers dried grain, whereas the thin stillage can be taken on toprovide the oil compositions.

One aspect provides an unrefined corn oil composition comprising havinga free fatty acid content of less than about 5 weight percent; amoisture content of from about 0.2 to about 1 weight percent; and analkali metal ion and/or alkaline metal ion content of greater than 10ppm up to about 1000 ppm. The unrefined corn oil has not been subjectedto a refining process. Such refining processes include alkali refiningand/or physical refining (i.e., distillation, deodorization, bleaching,etc.), and are used to lower the free fatty acid content, the moisturecontent, the insoluble content and/or the unsaponifiables content.

The free fatty acid content of the unrefined corn oil composition isless than about 5 weight percent. The oil composition described hereinhas a free fatty acid content level that can reduce the amount offront-end refining or processing for use in various applications. Insome embodiments, the free fatty acid content comprises at least onefatty acid selected from the group consisting of C₁₆ palmitic, C₁₈stearic, C₁₈₋₁ oleic, C₁₈₋₂ linoleic, and C₁₈₋₃ linolenic (where thenumber after the “-” reflects the number of sites of unsaturation). Insome embodiments, the free fatty acid content is less than 5 weightpercent. For example, in some embodiments, the free fatty acid contentis less than about 4 weight percent, or alternatively, less than about 3weight percent, or alternatively, less than about 2 weight percent, oralternatively, less than about 1 weight percent.

Maintaining low levels of moisture is advantageous as moisture canresult in the formation of free fatty acids. The unrefined corn oilcomposition may have a moisture content of less than about 1 weightpercent. The moisture in the present corn oil composition can comprisewater along with other soluble components, such as one or more alkaliand/or alkaline metal, and may further contain other soluble components,such as volatile material including hexane, ethanol, methanol, and thelike. The pH of the water that makes up the moisture content is, ingeneral, alkaline (i.e., >7) and comprises the one or more alkali and/oralkaline metals. In some embodiments, the moisture content of theunrefined corn oil composition is from about 0.2 to about 1 weightpercent, or alternatively, about or less than about 0.8 weight percent,or alternatively, about or less than about 0.6 weight percent, oralternatively, about or less than about 0.4 weight percent, oralternatively, about 0.2 weight percent. In certain embodiments, themetal ion concentration of the moisture content is about 2,000 ppm.Accordingly, an unrefined corn oil composition having from about 0.2 toabout 1 weight percent would have a metal ion concentration of fromabout 4 ppm to about 20 ppm. Typically, the moisture content of theunrefined corn oil composition is about 0.5 weight percent having ametal ion concentration of about 2000 ppm, resulting in an ionconcentration in the oil composition of about 10 ppm. In someembodiments, the unrefined corn oil composition has a metal ionconcentration of greater than about 0.4 ppm, or greater than about 0.5ppm, or greater than about 0.6 ppm, or greater than about 0.7 ppm, orgreater than about 0.8 ppm, or 20 ppm.

As is stated above, the moisture content is, in general, alkaline(i.e., >7). Accordingly, the water content in the oil comprises analkali metal ion and/or alkaline metal ion content of or greater thanabout 10 ppm. The alkali metal ion present in the composition can be anyalkali metal ion and/or any alkaline metal ion, and is in someembodiments, any combination of lithium (Li⁺), sodium (Na⁺), magnesium(Mg²⁺), potassium (K⁺) and/or calcium (Ca²⁺).

In some embodiments, the alkaline moisture content can comprise anorganic base, such as ammonia and/or ammonium ions. Accordingly, in oneembodiment, an unrefined corn oil composition comprises a free fattyacid content of less than about 5 weight percent; a moisture content offrom about 0.2 to about 1 weight percent; and an ammonia and/or ammoniumion content of greater than about 10 ppm, or from about 4 ppm to about20 ppm.

In some embodiments, the unrefined corn oil has an insoluble content ofless than about 1.5 weight percent. The insoluble content is notsolvated by the aqueous portion, the oil or the moisture within the oil,and can include material such as residual solid (e.g., corn fiber).

In some embodiments, the unrefined corn oil has an unsaponifiablescontent less than about 3 weight percent, or less than about 2 weightpercent, or less than about 1 weight percent. Unsaponifiable matter cansignificantly reduce the end product yields of the oil composition andcan, in turn, reduce end product yields of the methods disclosed herein.The unsaponifiables content of the oil does not form soaps when blendedwith a base, and includes any variety of possible non-triglyceridematerials that act as contaminants during biodiesel production.

The unrefined corn oil can further comprise various other oil solublecomponents. It is contemplated that the amount of such components wouldnot be so much that the unrefined corn oil composition would requirerefining prior to being used. Such components can include, for example,one or more of lutein, cis-lutein, zea-xanthin, alpha-cryptoxanthin,beta-cryptoxanthin, alpha-carotene, beta-carotene, cis-beta-carotene,alpha-tocopherol, beta-tocopherol, delta-tocopherol, orgamma-tocopherol, alpha-tocotrienol, beta-tocotrienol,gamma-tocotrienol, and/or delta-tocotrienol. In some embodiments, theunrefined corn oil composition has a tocopherol content less than about1 mg/g. In some embodiments, the unrefined corn oil composition has atocotrienol content less than about 1.3 mg/g. In some embodiments, theunrefined corn oil composition has a beta-carotene content greater thanabout 2 μg/g. Such components are known antioxidants and can thusprovide an oxidative stability to the unrefined corn oil composition.

The unrefined corn oil composition exhibits a high level of oxidativestability than corn oils prepared via conventional methods. This can bedue to any combination of factors, such as, the degree of saturation ofthe oil, the natural antioxidants, and the like, and can easily bedetermined using methods well known in the art. In some embodiments, theoxidative stability of the unrefined corn oil composition is greaterthan about 4 hours at a temperature of about 110° C. Further, theoxidative stability can be assessed using its peroxide value. In someembodiments, the unrefined corn oil composition exhibits a peroxidevalue of less than about 2 parts per million, or less than 1 part permillion.

Exemplary Methods of Providing Lower Alkyl Ester Containing OilCompositions from Fermentation Residue

One aspect is directed to a method for providing a vegetable oilcomposition from a vegetable, e.g., corn, fermentation residuecomprising the steps of: adjusting the pH of the vegetable, e.g., corn,fermentation residue to provide a vegetable, e.g., corn, oil layer andan aqueous layer: and separating the vegetable, e.g., corn oil layerfrom the aqueous layer to provide the vegetable, e.g., corn oilcomposition.

One aspect is directed to a method for providing a vegetable, e.g.,corn, oil composition from a vegetable, e.g., corn fermentation residuecomprising: separating the vegetable, e.g., corn, fermentation residueto provide an emulsion layer and a first aqueous layer; adjusting the pHof the emulsion layer to provide a vegetable, e.g., corn, oil layer anda second aqueous layer; and separating the vegetable, e.g., corn, oillayer from the second aqueous layer to provide the vegetable, e.g.,corn, oil composition.

In some embodiments, the vegetable, e.g., corn, fermentation residuecomprises whole stillage. In a fermentation process, the whole stillageis the remaining components of the fermenter after the ethanol has beendistilled. The whole stillage comprises a solid component and a liquidcomponent. The liquid component of the whole stillage is referred toherein as thin stillage. In one embodiment, the whole stillage can besubjected to further processing steps to produce thin stillage. Thinstillage can be recovered from the solid component of the whole stillageby natural phase separation and decanting, or can be accelerated usingmethods such as centrifugation. In one embodiment, the solid componentof the whole stillage can be subjected to drying to provide distillersdried grain and sold as an animal feed product. In some embodiments, thevegetable, e.g., corn, fermentation residue comprises thin stillage. Inone embodiment, moisture can be removed from the thin stillage to createa concentrated fermented product, herein referred to as syrup. Moisturecan be removed in a variety of ways such as, for example, throughevaporation under vacuum which, in turn, can prevent fouling.Accordingly, in some embodiments, the vegetable, e.g., corn,fermentation residue comprises syrup. In some embodiments, thevegetable, e.g., corn, fermentation residue has a moisture content ofbetween about 95% and about 60% weight percent. In some embodiments, thevegetable, e.g., corn fermentation residue has a moisture content ofabout 95%, or about 90%, or about 85%, or about 80%, or about 75%, orabout 70%, or about 65%, or about 60% weight percent.

The method optionally includes the step of separating the vegetable,e.g., corn, fermentation residue (whole stillage, thin stillage, orsyrup) to provide an emulsion layer and a first aqueous layer. The stepof separating can be accomplished by simply allowing the phaseseparation to occur over time and the oil layer decanted or by utilizingcentrifuge or a combination thereof, including, but not limited to, forexample, a press, extruder, a decanter centrifuge, a disk stackcentrifuge, a screen centrifuge or a combination thereof. In someembodiments, the separating does not comprise heating. In oneembodiment, a continuous flow at about 4000 g is maintained. One ofordinary skill in the art will appreciate that the speed or amount ofcentrifugal force applied will depend on various factors such as samplesize and may be adjusted appropriately depending on such factors.Suitable separators and centrifuges are available from variousmanufacturers.

In one embodiment, the resulting emulsion layer contains from about 20%w/w to about 70% w/w oil. In another embodiment, the emulsion layercontains from about 30% w/w to about 60% w/w oil. In yet anotherembodiment, the emulsion layer contains from about 40% w/w to about 50%w/w oil. The oil fraction may also comprise varying amounts of theoverall fermentation residue volume. In one embodiment, the emulsionlayer comprises about 20% w/w of the initial fermented product volume.

In one embodiment, the step of separating the vegetable, e.g., corn,fermentation residue is performed soon after initial production of theethanol in order to maintain oil composition quality and preventexposure to heat and oxygen, which are contributors to the formation offree fatty acids. The emulsion layer, which comprises the oilcomposition, is in one embodiment separated from the first aqueouslayer. All or a fraction of the first aqueous layer may be furtherprocessed or applied to solids such as, for example, distillers driedgrain.

In one embodiment, once separated from the first aqueous layer, the pHof the emulsion layer is adjusted such that the emulsion is sufficientlybroken, thus providing the oil composition and a second aqueous layer.The pH adjustment allows selective separation of higher quality oilwhile leaving the free fatty acids in an aqueous fraction by saponifyingthe fatty acids thus making them more water soluble. Thus, a portion ofthe free fatty acid is removed resulting in oil that contains low levelsof free fatty acid. The age of the fermented product and the organicacid content of the fermented product can affect the optimum pH forseparation, however, the oil fraction is treated with the highest pHpossible to reduce the overall free fatty acid content in the separatedoil without sacrificing oil quality. Typically, suitable pH's range fromabout 7.5 to about 10. The mixture of the free oil composition and oilfraction can be removed for further processing.

In another embodiment, the first aqueous layer is not removed from theemulsion layer but rather is subjected to base treatment to form the oillayer and the second aqueous layer which comprises both the firstaqueous layer and water resulting from breakage of the emulsion. The oillayer is then separated from the second aqueous layer. Accordingly, insome embodiments, the method comprises the steps of a) adjusting the pHof the vegetable fermentation residue to provide a corn oil layer and asecond aqueous layer: and b) separating the vegetable oil layer from thesecond aqueous layer to provide the vegetable oil composition. In someembodiments, the separating steps do not comprise heating.

In some embodiments, the pH of the emulsion layer is lowered by addingan acid. In one such embodiment, the pH can be adjusted downward byabout 1 pH unit, or about 2 pH units, or about 3 pH units. It iscontemplated that any inorganic or mineral acid can be used foradjusting the pH of the emulsion layer.

In some embodiments, the pH of the emulsion layer is raised by addingbase. In one such embodiment, the pH can be adjusted upward by about 1pH unit, or about 2 pH units, or about 3 pH units, or about 4 pH units,or about 5 pH units, or about 6 pH units. In some embodiments, the pH ofthe emulsion layer is less than about 4, or about 3.5, prior to the stepof adjusting the pH of the emulsion layer. It is contemplated that anyinorganic or mineral base can be used for adjusting the pH of theemulsion layer. Suitable bases include, but are not limited to, a baseselected from the group consisting of sodium hydroxide, sodiummethoxide, potassium hydroxide, calcium hydroxide, or spent alkali washsolution. In some embodiments, the base can be organic base, such asammonia. Efficient phase separation of the emulsion layer can beachieved by adjusting the pH of the emulsion layer to about 7.5 to about10, or from about 8 to about 9, or to a pH of about 8.2.

Once the emulsion has sufficiently broken, a corn oil layer and a secondaqueous layer are provided. The corn oil layer comprises the unrefinedcorn oil as disclosed herein.

In some cases, it may be that an interface layer is present between theoil layer and the aqueous layer, which is known in the art as a raglayer. The interface layer can comprise oil, water, phospholipids, freefatty acids, solids, etc. In some embodiments, the interface layer issubstantially removed from the oil layer with the aqueous layer.However, since the interface layer can comprise a significant amount ofoil, it may be advantageous to extract the oil from the interface layer.Accordingly, in some embodiments, the interface layer is kept with theoil layer and subjected to the pH adjustment step. The volume of theinterface layer can be decreased by about 50% or more by using a greatervolume of aqueous solution compared to the volume of the oil layer.Therefore, it may be advantageous to use a greater volume of aqueoussolution by adding water and/or using spent alkali wash solution. Suchmethods may provide an oil having a lower phospholipid concentration.

Accordingly, the unrefined vegetable, e.g., corn, oil as disclosedherein can be provided by separating the vegetable, e.g., corn, oillayer from the second aqueous layer. The step of separating thevegetable, e.g., corn, oil layer from the second aqueous layer can beaccomplished by simply allowing the phase separation to occur over timeand the oil layer decanted or by utilizing centrifuge or a combinationthereof, including, but not limited to, for example, a press, extruder,a decanter centrifuge, a disk stack centrifuge, a screen centrifuge or acombination thereof. In some embodiments, the separating does notcomprise heating. In one embodiment, a continuous flow at about 4000 gis maintained. One of ordinary skill in the art will appreciate that thespeed or amount of centrifugal force applied will depend on variousfactors such as sample size and may be adjusted appropriately dependingon such factors. Suitable separators and centrifuges are available fromvarious manufacturers.

In one embodiment, the second aqueous portion comprises 60% to 80%moisture, based on the total weight of the second aqueous portion. Inone embodiment, the second aqueous portion comprises 10% to 40% protein,based on the total weight of the second aqueous portion. In oneembodiment, the second aqueous portion comprises up to 50% oil, based onthe total weight of the second aqueous portion. The remainder of thesecond aqueous portion typically comprises starch, neutral detergentfiber, and the like. The second aqueous portion can be used to treatdistillers dried grain or other solids where an increased level of thesecomponents is desirable.

Uses

The oil composition can be used in a wide variety of applications. Suchexemplary applications include the areas of oleochemicals, feed (e.g.,animal feed) as well as oils suitable for human consumption, asphaltrejuvenation, performance grade (PG) asphalt enhancement and/orbio-diesel. Accordingly, one embodiment is a recycled asphaltcomposition or performance-grade composition comprising the unrefinedcorn oil composition as described herein which may decrease theviscosity of the resulting mixture and/or enhance the properties of thepavement made therefrom, e.g., enhanced resistance to cracking includingbut not limited to transverse cracking and age-induced surface cracking.

Oleochemicals include feedstock chemicals that are suitable forbio-diesel production (fatty acid methyl esters). Industrialoleochemicals are useful in the production of soaps, detergents, wireinsulation, industrial lubricants, leather treatments, cutting oils,mining agents for oil well drilling, ink removal, plastic stabilizers,ink and in rubber production. Other industrial applications includewaxes, shampoos, personal hygiene and food emulsifier or additiveproducts.

One embodiment is directed to a distillers dried grain comprising about4% or less fat. In some embodiments, the distillers dried grain furthercomprises about 30% protein.

In one embodiment, an asphalt binder blend composition is providedcontaining a corn oil composition disclosed herein and virgin asphalt oran asphalt mix composition containing a corn oil composition disclosedherein and asphalt material containing recycled asphalt. The addition ofa corn oil composition to virgin asphalt is referred to as “asphaltmodification”, whereas addition of a corn oil composition to an asphaltmaterial containing recycled asphalt, such as recycled asphalt pavement(RAP) or recycled asphalt shingles (RAS) which may contain aggregates,is referred to as “asphalt rejuvenation”. An asphalt binder blendcontaining virgin asphalt includes corn oil containing in one embodimentless than 5% free fatty acid (FFA) and between about, for example,greater that about 18% fatty acid lower alkyl esters, about 20 to about40% lower alkyl esters, or about 40 to about 80% or more lower alkylesters, by weight of the corn oil. An asphalt mix containing recycledasphalt includes in one embodiment corn oil containing in one embodimentless than 5% free fatty acid (FFA) and between about, for example,greater than about 18% fatty acid lower alkyl esters, about 20 to about40% fatty acid lower alkyl esters, or about 40 to about 80% or morefatty acid lower alkyl esters, by weight of the corn oil. The binderblend or asphalt mix may also contain other asphalt modifiers including,but not limited to, various petroleum fractions, polymers,polyphosphoric acid, lime, waxes, and/or antistrip agents. The range ofinclusion of DCO into asphalt is 1% to 25% by weight of the total binderblend.

The virgin or recycled asphalt can have a range of viscosity,penetration, stiffness, and viscoelastic properties that result inSuperpave performance grades (PGs) ranging from a high temperature of46° C. to 172° C. and a low temperature from −46° C. to 2° C. The finalPG of the resulting binder blend containing the asphalt, corn oilcomposition, and other modifiers can range in a high temperature from46° C. to 82° C. and a low temperature ranging from −46° C. to −10° C.The blending of a corn oil composition with recycled asphalt shouldincrease the ΔT_(c), which is decreased during the aging processindicating a loss in asphalt durability. The ΔT_(c) is defined as thedifference between the continuous stiffness temperature and continuousrelaxation temperature as measured by the bending beam rheometer (BBR)test (AASHTO T313).

For asphalt modification of virgin asphalt, the amount of a corn oilcomposition added in the final binder blend is dependent on theproperties of the virgin asphalt. A stiffer asphalt, defined as having alarge G* complex modulus (AASHTO T315), would require a higher inclusionof DCO or a DCO with higher fatty acid lower alkyl ester content. Forexample, a refining residue with a G* of 30.08 kPa at 64° C. requires10% inclusion of DCO to reduce it to 1.07 kPa at the same measuringtemperature. A virgin asphalt with a G* value of 1.21 kPa at 64° C. canbe blended with 4% inclusion of DCO to reduce the G* value to 0.57 kPaat the same temperature. As a result of the modification, the lowtemperature property of the binder blend is improved as well. The binderblend low temperature is determined by the stiffness and m-valuemeasured by the BBR test.

A corn oil composition can also be used in asphalt rejuvenation ofrecycled asphalt present in RAP and RAS. As asphalt is aged, the hinderbecomes oxidized and hardens decreasing the ΔT_(c) value indicating aloss of durability. In order to rejuvenate aged asphalt, a corn oilcomposition can be added to the recycled asphalt in order to increasethe ΔT_(c) value. In addition, inclusion of a corn oil compositionincreases the mix performance of RAP blends as observed as an increasein both low and intermediate cracking resistance without causing the mixto become susceptible to rutting. Typical inclusion of RAP in asphaltmixes may range from 1% to 50%. Inclusion of a corn oil composition inRAP containing asphalt mixes may range from 1% to 25% based upon theweight of the asphalt binder blend composition. For a hot mix, RAP orRAS can be rejuvenated by several different methods. A corn oilcomposition can be added onto the RAP or RAS stockpiles, added directlyinto the mix drum, or injected into the virgin asphalt. RAP/RAS can bepretreated by spraying the stream prior to its addition to the mix drum.A corn oil composition can also be added to virgin asphalt in storagetanks equipped with mixers or it can be added with an in-line staticmixer downstream prior to reaching the mix drum.

The pH level capable of providing an oil composition containing a lowlevel of free fatty acid can be determined (FIG. 3). First, an oilfraction in the form of an emulsion separated from fermented product maybe adjusted to the pH levels of 7.7, 7.9, 8.0, 8.1, 8.2, and 8.3. Thesamples may then be centrifuged to separate the oil composition and theoil composition was analyzed for free fatty acid content.

In summary, those samples tested at lower pH (i.e., below 8.0) exhibitedfree fatty acid contents above 3.5% w/w while those tested at a pH above8.1 exhibited a free fatty acid content of below 2% w/w.

TABLE 1 pH 7.7 7.9 8.0 8.1 8.2 8.3 Free Fatty Acids (percent) 3.5 2.22.0 2.2 2.0 1.8 Experiment 1 Free Fatty Acids (percent) 4.8 3.5 3.1 2.22.0 1.8 Experiment 2

A series of oil fractions, in the form of emulsions samples previouslyseparated by a first application of a centrifugal force were treatedwith NaOH to adjust the pH to various levels as shown in Table 2. Eachsample contained the same amount of oil before adjusting the pH. Afteradjusting the pH to the targeted value, the volume of free oil wasmeasured.

A pH at about 8.2 may result in the highest value of free oil volume.The volume of free oil was shown to increase up to this value and thendeteriorate thereafter. Thus, an optimum pH for separation exists foreach oil fraction sample.

TABLE 2 pH 7.0 7.4 7.8 8.0 8.2 8.4 8.8 9 2 10.0 Free Fatty Acids(percent) 1.0 30 42 45 60 48 50 45 43 Experiment 1

Experiments may be conducted to demonstrate that the combination ofadjusting the pH and applying a centrifugal force resulted in (a) higherquality corn oil compositions and (b) higher corn oil composition yieldcompared to those oil compositions obtained upon application of acentrifugal force alone. The free fatty acid content may be shown to bereduced by up to 3% by adjusting the pH in combination with centrifugalforce as opposed to centrifugal force alone. The yield of separated oilcomposition may be increased by 140%. The experiment was run for about30 days, and includes 3 daily samples.

A compositional analysis of the products obtained from one embodiment ofthe system may be performed. The syrup fraction obtained from theethanol production process may be centrifuged to separate into a lightfraction (emulsified oil) and a heavy fraction (stickwater). The syrupobtained may be mostly free of oil. The heavy fraction may be returnedto the normal process to be further evaporated and added to wet cake anddried.

The pH of the light fraction may be raised to approximately 8.2 from apH of approximately 3.5. The pH adjusted emulsified material may be fedto a second centrifuge step. The heavy fraction (soapstock) from thesecond centrifuge step may be high in soaps and proteins and may bemixed with the stickwater and added to the wet cake and dried. The lightfraction from the second centrifuge may be oil. The oil may exhibit ahigh quality and low free fatty acid content, insolubles, moisture,phospholipids and unsaponifiables. The oil may be used with or withoutfurther refining. The distillers dried grains composition projected toresult from the combination of wet cake, soapstock, and low fat syrupmay exhibit lower fat and higher protein than typical for distillersdried grain.

TABLE 3 Fat Protein Moisture Other (per- (per- (per- (per- cent) cent)cent) cent)*** Starting Material* 5.4 4.1 80 10 First Light Fraction 353.6 55 6.8 (Emulsified Oil)* First Heavy Fraction 3.5 4.2 83 10(Stickwater)* Second Light Fraction 98 0.0 0.8 1.6 (Oil Composition)*Second Heavy Fraction 5.5 5.9 77 11 (Soapstock)* Low Fat DDGS** 4.0 308.7 57 *= Sampled, **= Projected, ***= Includes fiber, ash, starch, etc.

In a conventional dry-grind ethanol process, whole corn is ground to aflour, mixed with water and cooked at a high temperature to gelatinizethe starch and to make it more available for subsequent liquefaction andsaccharification by enzymes. The cooked mash is then cooled tofacilitate fermentation of the sugars into ethanol. The resulting beerincludes soluble and insoluble components, such as proteins, oil, fiber,residual starch and glycerol. The beer is separated into ethanol andwhole stillage in distillation. The whole stillage can be dewatered toproduce wet cake by removing a thin stillage component bycentrifugation. The oil partitions fairly equally, by weight, betweenthin stillage and the wet cake. Thin stillage is typically furtherevaporated into syrup, which can be added back onto the wet cake duringa drying process that produces distillers dried grains with solubles(i.e., DOGS). Corn oil can be recovered from the syrup by a simplecentrifuging step, as described for example in U.S. Pat. No. 7,601,858.

Some dry-grind ethanol processing facilities utilize a modified drygrind process known as raw starch ethanol production. In thesefacilities, the corn is ground to fine flour, mixed with water andenzymes, and fermented to ethanol-containing beer in a simultaneoussaccharification and fermentation reaction. The rest of the raw starchprocess is similar to the conventional process. However, in the rawstarch process the oil cannot be separated from the syrup by a simplecentrifugation step, but requires an additional treatment step (pHadjustment) and a second centrifugation step to recover the oil.Overall, raw starch ethanol production requires less energy and coolingwater.

Oil extracted from corn DDGS using solvents, and oil extractedcentrifugally from thin stillage have similar, or slightly lowerconcentrations of tocopherols than corn germ oil, but have higherconcentrations of phytosterols, tocotrienols, and steryl ferulates, thancorn germ oil.

The following provides exemplary methods and analyses of vegetable oilcompositions.

Materials and Methods Chemicals

Dry chemicals (ACS grade or better) were obtained from Sigma-Supelco(St. Louis, Mo.) unless otherwise noted in referenced methods. Solventswere HPLC grade and were obtained from Fisher (Fairlawn, N.J.).

Oils

The five oils that were characterized included hexane Soxhlet extractsof corn germ (CG) and DDGS (DDGS), and three oils that werecentrifugally extracted from dry grind ethanol production facilities(CS-1, CS-2, CS-3). The corn germ was obtained from an ethanolproduction facility that operates a dry fractionation process where thecorn kernels are separated into germ, fiber, and endosperm fractionsprior to fermentation. Corn DOGS was obtained from a raw starch ethanolproduction facility operated by POET, LLC (Sioux Falls, S.D.). CG andDDGS were extracted overnight (about 20 hr) by Soxhlet extraction usinghexane. Four parallel Soxhlet extractors with about 100 g/thimble wereused several days in a row and the extracts were combined to obtainenough oil from the germ and DDGS for analyses and storage studies.Hexane was removed by rotary evaporation at 40° C., oil was then stirredfor 4 hours under a high vacuum to remove any excess hexane, after whichthe oil was put into several amber bottles, topped with argon to preventlipid oxidation, and frozen at −20° C. until used for analyses. CS-1 wasobtained from a conventional dry grind ethanol plant. CS-2 and CS-3 wereobtained from two different production runs from a raw starch ethanolproduction facility operated by POET. CS-1, CS-2, and CS-3 were shippedovernight, on dry ice, to the research location, and immediatelytransferred to glass bottles, topped with argon, and frozen (−20° C.)until used for analyses.

Oil Analysis Acid Value

Acid Value was determined by titration using AOCS official method Cd3d-63 (AOCS, 1998). The acid value was used to calculate the percentfree fatty acids (FFA) as percent oleic acid by dividing the acid valueby 1.99 as stated in the method. Each oil was analyzed in triplicate forAcid Value and the mean is reported,

Fatty Acid Composition and Iodine Value

Oil triacylglycerols were transesterified using the method described byIchihara (1996). Fatty acid methyl esters were analyzed in triplicate byGC. The Iodine Values were calculated based on the fatty acidcomposition according to the AOCS Method Cd 1c-85 (AOCS, 1998).

Tocopherols, Phytosterols, and Steryl Ferulate Analysis

The contents of tocopherols, tocotrienols, and steryl ferulates wereanalyzed in triplicate in the crude oils by HPLC with a combination ofUV and fluorescence detection as previously described (Winkler et al.,2007). In order to analyze total phytosterol content and composition,the oils were saponified, and the phytosterols were extracted andderivatized as previously described (Winkler et al., 2007). Phytosterolswere quantitated by GC as described by Winkler and Vaughn (2009). Theidentity of phytosterol peaks was confirmed by GC-MS analysis performedon an Agilent (Santa Clara, Calif., USA) 6890 GC-MS equipped with aHP-5MS capillary column (30 m 9 0.25 mm 9 0.25 lm), a 5973 massselective detector, and an 7683 autosampler. The transfer line from GCto the MSD was set to 280° C., The injector and oven temperatureprograms were the same as described above for the GC-FID instrument. MSDparameters were as follows: scan mode, 50-600 amu, ionizing voltage, 70eV, and EM voltage, 1,823 V. Mass spectral identification was performedusing the Wiley MS database combined with comparison to literaturevalues for relative RT (compared to p-sitosterol) and mass spectra(Beveridge et al., 2002).

Carotenoid Analysis

Carotenoid analysis and quantitation were conducted by HPLC as describedby Winkler and Vaughn (2009).

Oxidative Stability Index

The OSI at 110° C. was determined in triplicate following the AOCSOfficial Method Cd 12b-92 (AOCS, 1998). A Metrohm (Herisau, Switzerland)743 Rancimat with software control automatically controlled air flow andtemperature and calculated the OSI values based on induction time.

Accelerated Storage Study

The study protocol followed AOCS Recommended Practice Cg 5-97 (AOCS,1998). Oil samples (5 g) were weighed into 40-ml amber glass vials whichwere loosely capped. For each treatment and day, triplicate vials wereprepared. Vials were stored in completely randomized order in a darkoven held at 40±1° C. For each oil, three vials were removed on days onethrough six and on day eight. CG oil samples were also removed on days10 and 12. However, as the study progressed, it was determined that theDDGS and CS-2 oils were oxidizing more slowly than the CG oil, sosamples were removed on days 12 and 14 order to extend their storage bytwo more days. Upon removal from the oven, vials were immediately toppedwith argon, tightly capped, and frozen (−20° C.) until analysis.Analyses were conducted either on the same day or within 2 days ofremoval from the oven. Peroxide values were determined using the methoddescribed by Shantha and Decker (1994). Each oil replicate from thestorage studies was analyzed in duplicate. Hexanal in the oil headspaceof each replicate was quantified in duplicate by solid-phasemicroextraction (SPME) and GC analysis as described by Winkler andVaughn (2009),

Room Temperature Storage Study

CS-2 oil was placed into three, 4 L amber bottles. Each bottle wasfilled to the same volume level of 3.4 L. The amount of headspace abovethe oil samples amounted to 0.9 L. Bottles were tightly capped andstored in the dark at 20° C.±3° C., the temperature was monitored dailyand the high and low temperature was recorded. Samples were taken once aweek for 13 weeks. To sample, bottles were first gently shaken for 30seconds to mix the contents. Then a glass pipet was inserted into thecenter of the bottle and 5 ml oil was taken and placed into a screw capvial, covered with argon, and frozen (−20° C.) until analysis. Peroxidevalue and headspace analysis of hexanal were performed on the oilsamples as described above, and were typically run on the same day orwithin 1-2 days of sampling.

Fatty Acid Composition and Free Fatty Acids

The fatty acid compositions (Table 4) of all five oils were typical forcorn oil. The Iodine Values ranged from 122.4 to 124.3. These resultsconcur with other reports that the fatty acid composition of oilextracted from DDGS and thin stillage are similar to corn oil. The twooils (CS-1 and CS-2) that were centrifugally extracted from syrup fromthe raw starch ethanol production facilities had the lowest % FFA (2.03%and 2.48%, respectively). The oil recovered by centrifugation of syrupfrom the traditional dry grind ethanol production plant had the highestAcid Value, with 10.1% FFA. Other studies have reported FFA content ofoil recovered by centrifugation of thin stillage ranging from11.2-16.4%. These results indicate that the elimination of the cookingstep in the raw starch process reduces the production of FFA. The oilextracted from DDGS using hexane had the second highest acid value(7.42% FFA). Winkler-Moser and Vaughn (2009) reported FFA content of6.8% (w/w) in hexane Soxhlet extracted DOGS oil, while Moreau et al.(2010) reported FFA content ranging from 8-12% in DOGS that wasextracted with hexane using accelerated solvent extraction. FFA contentof DDGS extracts has been shown to vary widely depending on theextraction method and conditions and on the solvent used. The DOGS usedin this study also came from a raw starch ethanol plant, so it might beexpected to have lower FFA. However, high temperatures used to dry thewet grains may have contributed to the increase in FFA. In oneexperiment. Moreau et al. (2010) demonstrated that oil extracted fromthin stillage and distillers dried grains (prior to mixing the grainswith the syrup) had high FFA content that carried through to the DDGS.The FFA content of hexane extracted corn germ was 3.8%, which isslightly higher than the average of 2.5% FFA typically found in crudecorn germ oil. For biodiesel production, oil with an Acid Value greaterthan one requires pretreatment because the free fatty acids form soapsduring base-catalyzed esterification, which interfere with theseparation of the glycerol from the fatty acid methyl esters. Thus,crude oils with lower free fatty acids will have lower oil loss due tothe pre-treatment. Free fatty acids decrease the oxidative stability ofoils and can also precipitate at ambient temperatures, both of whichcould negatively impact fuel performance.

TABLE 4 Acid value, fatty acid composition, and calculated Iodine Valueof oils extracted from corn germ (CG), distillers dried grains withsoluble (DDGS), and centrifugally extracted thin stillage syrup (CS-1,CS-2, CS-3) CG DDGS CS-1 CS-2 CS-3 Acid Value 10.7 ± 20.8 ± 28.3 ± 5.70± 6.88 ± (mg KOH/g) 0.07 0.36 0.32 0.13 0.09 FFA 3.80 ± 7.42 ± 10.1 ±2.03 ± 2.48 ± (% oleic acid) 0.03 0.13 0.11 0.05 0.05 Fatty AcidComposition (%) 16:0 13.1 12.9 11.5 12.2 12.9 16:1 0.0 0.1 0.1 0.1 0.118:0 1.5 1.8 1.7 1.8 1.5 18:1 29.2 28.1 29.3 28.3 27.5 18:2 55.0 55.555.6 55.3 55.9 20:0 0.2 0.3 0.3 0.4 0.3 18:3 1.0 1.2 1.17 1.2 1.2 20:10.0 0.0 0.2 0.3 0.2 Calculated Iodine 122.4 123.1 124.3 123.7 124.1Value

Content and Composition of Tocopherols, Tocotrienols, and Carotenoids

Tocopherols are common in vegetable oils and are the primaryantioxidants protecting most oils. With corn and other plants, thetocopherol and tocotrienol content will vary based upon factorsincluding hybrid, growth conditions, post-harvesting and processingconditions, as well as the type of solvent used for extraction.Therefore, in this study little can be inferred about how processingpractices affected tocopherol levels since each production facility andeven each production run will have started with different batches ofwhole corn. Gamma- and alpha-tocopherol were the most prominenthomologues detected in all five oils (Table 5), along with a smallamount of delta-tocopherol, which is the typical tocopherol profile forcorn oil. CG oil had the highest total concentration of tocopherols(1433.6 μg/g oil) followed by the hexane extracted DDGS (1104.2). Thelevels in the DOGS oil are similar to what was previously reported inhexane extracted DOGS from a conventional dry grind production facility.Tocopherols in corn are localized in the germ portion of the kernel, sothe rest of the corn kernel contributes little to the tocopherolcontent. CS-1, CS-2, and CS-3 were all lower in alpha-tocopherolcompared to CG and DOGS oils, but were similar to levels reported in oilextracted centrifugally from thin stillaae (Moreau et al., 2010).

TABLE 5 Content of tocols and carotenoids, and the oxidative stabilityindex (OSI) at 110° C., for oils extracted from corn germ (CG),distillers dried grains with solubles (DDGS), and centrifugallyextracted thin stillage syrup (CS-1, CS-2, CS-3) CG DDGS CS-1 CS-2 CS-3Total Tocopherols 1433.6 1104.2 1056.9 931.3 783.4 (μg/g)Alpha-tocopherol 213.8 295.6 164.5 160.4 123.2 Gamma-tocopherol 1185.4760.8 852.7 742.0 640.0 Delta-tocopherol 34.3 47.8 39.7 28.8 20.2 TotalTocotrienols 235.6 1762.3 1419.6 1224.4 1175.2 (μg/g) Alpha-tocotrienol21.9 471.9 328.5 243.6 269.4 Gamma-tocotrienol 165.6 1210.0 1063.6 963.4880 Delta-tocotrienol 48.1 80.3 27.5 17.3 25.8 Total Carotenoids 1.3375.02 129.48 61.1 85.0 (μg/g) Lutein 0.37 46.69 75.69 38.13 53.7Zeaxanthin 0.4 24.16 45.58 16.78 23.7 Beta-cryptoxanthin 0.56 3.31 7.354.12 5.1 Beta-carotene ND^(a) 0.86 0.86 2.07 2.5 OSI (hr) 3.91 6.62 4.454.52 5.27 ^(a)Not detected

Tocotrienols are common in rice bran oil and palm oil, but are notabundant in most commercial vegetable oils. Their antioxidant activityis similar to tocopherols in bulk oil systems, but they also appear tohave hypocholesterolemic, anti-cancer, and neuroprotective properties.The post-fermentation corn oils (DOGS, CS-1, CS-2, and CS-3) were higherin tocotrienol concentration compared to CG oil, because tocotrienolsare found in the endosperm fractions, which are mostly removed duringthe fractionation of corn germ. Thus, despite having lower tocopherolconcentration, all of the post-fermentation oils were higher in totaltocol concentration compared to the CG oil.

The post-fermentation corn oils were much higher in carotenoids than theextracted corn germ oil as well. However, the concentration ofcarotenoids was substantially lower than the tocols in five oils (Table5). As with tocotrienols, carotenoids are localized to the endospermfraction of corn kernels. The main carotenoids in the oils were luteinand zeaxanthin, as well as lower quantities of beta-cryptoxanthin andbeta-carotene. Carotenoid content and composition were similar toamounts found in DOGS oil in a previous study, however. Moreau et al.(2010) reported carotenoid content in centrifugally extracted thinstillage oil ranging from 295 to 405 μg/g oil. Carotenoids aresubstantially affected by corn hybrid, which may explain thediscrepancy. Beta-carotene and beta-cryptoxanthin are both precursors toVitamin A, while lutein and zeaxanthin are both protective againstage-related macular degeneration and cataracts. Carotenoids have alsobeen shown to have a number of beneficial physiological actions otherthan Vitamin A activity, including antioxidant activity, enhanced immuneresponse, and chemoprotective activity against several types of cancer.

Content and Composition of Phytosterols

The content of total phytosterols in the three oils ranged from 1.5-2.0%(w/w) (Table 6). The post-fermentation corn oils were higher in totalphytosterols compared to the CG oil because they include phytosterolsand ferulate phytosterol esters from the bran and pericarp, in additionto the phytosterols from the germ portion of the corn kernel. Thephytosterol composition is also different between CG oil and thepost-fermentation corn oils. DDGS and CS-1, CS-2, and CS-3 oils hadsimilar concentrations of the common phytosterols campesterol,stigmasterol, and sitosterol compared to CG oil. However, they had amuch higher concentration of the two saturated phytosterols(phytostanols), campestanol and sitostanol. The high content of thesephytostanols is due to their preferential esterification, in corn, tosteryl ferulates, the contents of which are also shown in Table 6.Steryl ferulates are found in the inner pericarp of corn and othergrains. The presence of a small amount of these compounds in the corngerm oil indicates that there may have been some contamination of thegerm by some inner pericarp tissue, as it has been established thatthese compounds are unique to the aleurone layer of the pericarp.Phytosterols are highly valued as ingredients in functional foods due totheir ability to lower blood cholesterol by blocking re-adsorption ofcholesterol from the gut. Steryl ferulates have been shown to retain thecholesterol lowering ability of phytosterols, and also have antioxidantactivity due to the ferulic acid moiety.

TABLE 6 Content and compositions of phytosterols in oils extracted fromcorn germ (CG), distillers dried grains with solubles (DDGS), andcentrifugally extracted thin stillage syrup (CS-1, CS-2, CS-3). CG DDGSCS-1 CS-2 CS-3 mg/g %^(a) mg/g % mg/g % mg/g % mg/g % Total 14.9  21.718.7  20.1 20.2 Phytosterols Campesterol 3.08 20.7 2.97 13.7 2.74 14.72.74 13.6 3.0 14.7 Campestanol 0.25 1.7 1.35 6.2 1.40 7.5 1.30 6.5 1.46.7 Stigmasterol 0.98 6.6 1.10 5.1 0.76 4.1 0.91 4.5 0.89 4.4 Sitosterol9.04 60.9 10.3 47.5 8.77 46.9 9.36 46.5 9.3 46.1 Sitostanol 0.66 4.43.72 17.2 3.59 19.2 3.45 17.2 3.2 16.0 Avenasterol 0.54 3.7 0.93 4.30.86 4.6 0.94 4.7 1.0 5.2 Cycloartenol 0.28 1.9 0.71 3.2 0.59 3.2 0.743.7 0.73 3.6 24-methylene ND^(b) 0 0.30 1.4 ND 0 0.34 1.7 0.30 1.5cycloartanol Citrostadienol ND  0 0.31 1.4 ND 0 0.31 1.6 0.36 1.8 Steryl0.58 3.9 3.42 15.7 3.15 16.8 3.38 16.8 3.35 16.6 Ferulates (mg/g)^(a)The weight percentage of total phytosterols ^(b)Not detected

Oxidative Stability Index (OSI)

The oxidative stability of oils are affected by many factors, includingfatty acid composition, concentration and stability of antioxidants inthe oil, and the presence of prooxidant compounds, such as free fattyacids, lipid peroxides, or prooxidant metals. The Rancimat is anaccelerated test (taking several hours to a day, depending on the oiland test temperature) used to establish the relative oxidative stabilityof oils, as measured by the induction time (called the oxidativestability index, OSI) for an oil to begin oxidizing under controlledtemperature and air flow conditions. The OSI of the CG oil was lowest,while DDGS oil had the highest stability (Table 5), which corresponds tothe lowest and the highest concentration of antioxidant tocopherols.CS-1 had a slightly lower OSI than CS-2 and CS-3 despite having a higherconcentration of tocols; this may be explained by its higher content ofFFA and higher initial peroxide value.

Conclusions

This Example compared the composition and oxidative stability of oilsextracted from corn germ, corn distillers dried grains, and from thinstillage from a conventional dry grind ethanol production facility aswell as from a raw starch ethanol production facility. The fatty acidcompositions of all five oils were typical for corn oil. Oil extractedfrom thin stillage in a raw starch production facility has lower FFAthan from thin stillage from a conventional dry grind ethanol productionfacility. This is likely due to lower processing temperatures used inthe raw starch process where the cooking stage is eliminated. All of thepost-fermentation oils had higher concentrations of tocotrienols,carotenoids, phytosterols, and ferulate phytosterol esters compared tothe corn germ oil. The increased concentrations of the antioxidanttocotrienols carotenoids, and steryl ferulates are likely responsiblefor their increased stability compared to corn germ oil.

Other Exemplary Embodiments

Also provided is a corn oil composition comprising unrefined corn oilhaving an ethyl ester content that is greater than 7 weight percent,e.g., greater than 18 weight percent; and optionally a moisture contentof from about 0.02 to about 1 weight percent and/or an alkali metal ionand/or alkaline metal ion content of greater than 10 ppm up to about1000 ppm. In one embodiment, the unrefined corn oil has a free fattyacid content of less than about 5 weight percent. In one embodiment, theunrefined corn oil has an ethyl ester content that is greater than 30weight percent. In one embodiment, the unrefined corn oil has aninsoluble content of less than about 1.5 weight percent. In oneembodiment, the unrefined corn oil has a free fatty acid content of lessthan about 3 or less than about 2 weight percent. In one embodiment, theunrefined corn oil has a peroxide value of less than about 2 pails permillion. The corn oil composition may include a lutein content of atleast 50 mcg/g, a zeaxanthin content of at least 30 mcg/g, acis-lutein/zeaxanthin content of at least 10 mcg/g, analpha-cryptoxanthin content of at least 5 mcg/g, a beta-cryptoxanthincontent of at least 5 mcg/g, an alpha-carotene content of at least 0.5mcg/g, a beta-carotene content of at least 1 mcg/g, a cis-beta-carotenecontent of at least 0.1 mcg/g, an alpha-tocopherol content of at least50 mcg/g, a beta-tocopherol content of at least 2 mcg/g, agamma-tocopherol content of at least 300 mcg/g, a delta-tocopherolcontent of at least 15 mcg/g, an alpha-tocotrienol content of at least50 mcg/g, a beta-tocotrienol content of at least 5 mcg/g, agamma-tocotrienol content of at least 80 mcg/g, a delta-tocotrienolcontent of at least 5 mcg/g, or any combination thereof.

In one embodiment, to prepare a vegetable oil composition, fermentationis employed. For example, a method for enhancing vegetable oilproperties from ground plant material subjected to fermentation isprovided. The method includes providing an aqueous compositioncomprising ground plant material, e.g., seeds, sized such that more than50% of the ground material passes through a 0.5 mm screen, a fungal acidamylase and a glucoamylase under conditions which produce glucoseincluding a pH of from 3 to 6, a temperature of from about 25° C. toabout 40° C. and a solids content in said composition of from about 20to 50 weight percent; and fermenting the glucose in the presence of ayeast and a composition comprising an esterase under conditions whichproduce ethanol and vegetable oil having an ethyl ester content that isgreater than 18% w/w based on the total weight of the oil composition,wherein said conditions include a pH of from about 3 to 6 andmaintaining a glucose concentration in the aqueous composition of lessthan about 2 weight percent after 12 hours of saccharification andfermentation, wherein said method produces at least 18 volume percentethanol. In one embodiment, the vegetable oil has a free fatty acidcontent of no greater than 5% w/w based on the total weight of thecomposition. In one embodiment, the ethyl ester content is greater thanabout 20% w/w in the total weight of the oil composition. In oneembodiment, the ethyl ester content is greater than about 30% w/w in thetotal weight of the composition. In one embodiment, the ethyl estercontent is greater than about 50% w/w in the total weight of the oilcomposition. In one embodiment, the ethyl ester content is greater thanabout 60% w/w in the total weight of the oil composition. In oneembodiment, the esterase is a plant or a fungal esterase. In oneembodiment, the esterase is a carboxylic ester hydrolase (EC 3.1.1.3).In one embodiment, the esterase is a lipase. In one embodiment, theesterase is in an amount that is at least 0.01% w/w of the weight ofplant fat in the aqueous composition prior to fermentation. In oneembodiment, the esterase is in an amount that is at least 0.04% w/w ofthe weight of plant fat in the aqueous composition prior tofermentation. In one embodiment, the esterase is in an amount that is atleast 0.4% w/w of the weight of plant fat in the aqueous compositionprior to fermentation. In one embodiment, during the production ofethanol, the pH is maintained at 3-4.5 during the first half of the fillcycle and at 4.5-6.0 during the second half of the fill cycle. In oneembodiment, glucose is produced at a temperature of from about 30° C. toabout 35° C., a solids content in said composition of from about 25 to45 weight percent, an amount of said fungal acid amylase which rangesfrom about 0.1 to about 10 fungal acid amylase units per gram of saiddry solids, and an amount of said glucoamylase to dry solids in saidcomposition which ranges from about 0.5 to about 6 glucoamylase unitsper gram of said dry solids. In one embodiment, the glucose is fermentedunder conditions comprising an initial temperature of about 35° C. whichtemperature is decreased during fermentation to a temperature of about30° C., and maintaining a glucose concentration in the aqueouscomposition of less than about 1 weight percent after 12 hours ofsaccharification and fermentation, wherein the production of glucose andthe fermentation of glucose to ethanol is conducted simultaneously.

In one embodiment, to prepare a vegetable oil composition, fermentationis employed. For example, a method for enhancing vegetable oilproperties from ground plant material subjected to fermentation isprovided. The method includes providing an aqueous compositioncomprising ground plant material, e.g., seeds, sized such that more than50% of the ground material passes through a 0.5 mm screen, a fungal acidamylase and a glucoamylase under conditions which produce glucoseincluding a temperature of from about 25° C. to about 40° C. and asolids content in said composition of from about 20 to 50 weightpercent; and fermenting the glucose in the presence of a yeast and acomposition comprising an esterase under conditions which produceethanol and vegetable oil having an ethyl ester content that is greaterthan 18% w/w based on the total weight of the oil composition. In oneembodiment, the vegetable oil has a free fatty acid content of nogreater than 5% w/w based on the total weight of the composition. In oneembodiment, the ethyl ester content is greater than about 20% w/w in thetotal weight of the oil composition. In one embodiment, the ethyl estercontent is greater than about 30% w/w in the total weight of thecomposition. In one embodiment, the ethyl ester content is greater thanabout 50% w/w in the total weight of the oil composition. In oneembodiment, the ethyl ester content is greater than about 60% w/w in thetotal weight of the oil composition. In one embodiment, the esterase isa plant or a fungal esterase. In one embodiment; the esterase is acarboxylic ester hydrolase (EC 3.1.1.3). In one embodiment; the esteraseis a lipase. In one embodiment, the esterase is added when fermentationis initiated, after fermentation is initiated; when fermentation iscomplete, or any combination thereof. In one embodiment, the esterase isin an amount that is at least 0.01% w/w of the weight of plant fat inthe aqueous composition prior to fermentation. In one embodiment, theesterase is in an amount that is at least 0.04% w/w of the weight ofplant fat in the aqueous composition prior to fermentation. In oneembodiment, the esterase is in an amount that is at least 0.4% w/w ofthe weight of plant fat in the aqueous composition prior tofermentation. In one embodiment, glucose is produced at a temperature offrom about 30° C. to about 35° C., a solids content in said compositionof from about 25 to 45 weight percent, an amount of said fungal acidamylase which ranges from about 0.1 to about 10 fungal acid amylaseunits per gram of said dry solids, and an amount of said glucoamylase todry solids in said composition which ranges from about 0.5 to about 6glucoamylase units per gram of said dry solids. In one embodiment, theglucose is fermented under conditions comprising an initial temperatureof about 35° C. which temperature is decreased during fermentation to atemperature of about 30° C.

In one embodiment, a method for providing a corn oil composition withenhanced levels of, in one embodiment, ethyl ester, includes obtaining afirst aqueous layer from a corn fermentation residue; adjusting the pHof the first aqueous layer to provide a corn oil layer and a secondaqueous layer: and separating the corn oil layer from the second aqueouslayer to provide the corn oil composition having a free fatty acidcontent of less than about 2% or less than about 5% and has at least 10%w/w ethyl ester. In one embodiment, the first aqueous layer has amoisture content of between about 95% and about 60%. In one embodiment,the first aqueous layer comprises thin stillage. In one embodiment, themethod further comprises evaporating the thin stillage prior to the stepof adjusting the pH of the first aqueous layer. In one embodiment, thefirst aqueous layer comprises syrup. In one embodiment, adjusting the pHcomprises adding a base. In one embodiment, adjusting the pH comprisesadding a base selected from the group consisting of sodium hydroxide,potassium hydroxide, calcium hydroxide, or spent alkali wash solution.In one embodiment, the pH of the first aqueous layer is less than about4 prior to the step of adjusting the pH of the first aqueous layer. Inone embodiment, the pH of the first aqueous layer is about 3.5 prior tothe step of adjusting the pH of the first aqueous layer. In oneembodiment, the pH of the first aqueous layer is from about 7.5 to about10 after adjusting the pH of the first aqueous layer. In one embodiment,the pH of the first aqueous layer is from about 8 to about 9 afteradjusting the pH of the first aqueous layer. In one embodiment, the pHof the first aqueous layer is about 8.2 after adjusting the pH of thefirst aqueous layer. In one embodiment, obtaining the first aqueouslayer from the corn fermentation residue comprises centrifuging. In oneembodiment, obtaining the first aqueous layer from the corn fermentationresidue comprises a) separating the first aqueous layer into a waterlayer and an emulsion layer; and b) adjusting the pH of the emulsionlayer to provide a corn oil layer and a second aqueous layer. In oneembodiment, obtaining the first aqueous layer from the corn fermentationresidue to provide an emulsion layer and a first aqueous layer comprisescentrifuging. In one embodiment, separating the corn oil layer from thesecond aqueous layer comprises centrifuging. In one embodiment, the cornoil layer comprises a free fatty acid content of less than about 2weight percent. In one embodiment, the corn oil layer comprises amoisture content of from about 0.2 to about 1 weight percent. In oneembodiment, the corn oil layer comprises an alkali metal ion and/oralkaline metal ion content of greater than 10 parts per million. In oneembodiment, the corn oil layer has an insoluble content of less thanabout 1.5 weight percent. In one embodiment, the corn oil layer exhibitsa peroxide value of less than about 2 parts per million. In oneembodiment, the corn oil layer exhibits an oxidative stability ofgreater than about 4 hours at a temperature of about 110° C.

Also provided is a method for making a paving composition. The methodincludes combining a plurality of solids (aggregate) with an asphaltbinder blend composition to produce a paving composition, wherein theasphalt binder blend composition comprises bitumen and a corn oilcomposition having: an ethyl ester content of greater than 7%, e.g.,greater than about 18%, w/w based on the total weight of the oilcomposition; and optionally an iodine value of not greater than 125and/or a combined moisture and insoluble content of no greater than 1.5%w/w based on the total weight of the composition; and also optionally afurther component selected from the group consisting of: a luteincontent of at least 50 mcg/g, a cis-lutein/zeaxanthin content of atleast 10 mcg/g, an alpha-cryptoxanthin content of at least 5 mcg/g, abeta-cryptoxanthin content of at least 5 mcg/g, an alpha-carotenecontent of at least 0.5 mcg/g, and a cis-beta-carotene content of atleast 0.1 mcg/g. In one embodiment, the plurality of solids comprisessand, gravel, crushed stone, crushed concrete, crushed glass, industrialslag, or any mixture thereof.

In addition, an asphalt mix composition is provided comprising: recycledasphalt and a vegetable oil composition having an ethyl ester contentthat is greater than about 7% such as greater than about 18% w/w basedon the total weight of the oil composition; and optionally an iodinevalue of not greater than 125 and/or a combined moisture and insolublecontent of no greater than 1.5% w/w based on the total weight of thecomposition; and also optionally a further component selected from thegroup consisting of: a lutein content of at least 50 mcg/g, acis-lutein/zeaxanthin content of at least 10 mcg/g, analpha-cryptoxanthin content of at least 5 mcg/g, a beta-cryptoxanthincontent of at least 5 mcg/g, an alpha-carotene content of at least 0.5mcg/g, and a cis-beta-carotene content of at least 0.1 mcg/g. In oneembodiment, the vegetable oil is about 1 wt % to about 25 wt % based onweight of the asphalt binder composition or the asphalt binder blendcomposition. In one embodiment, the vegetable oil is about 0.5 wt % toabout 25 wt ° j % based on weight of the asphalt binder composition orthe asphalt binder blend composition. In one embodiment, the asphalt mixcomposition comprises virgin asphalt and recycled asphalt.

The invention will be further described with respect to the followingexamples.

Example 1 Materials and Methods

A simultaneous saccharification and fermentation (SSF) process isemployed, where starch-based feedstocks such as corn (maize), sorghum(milo), and/or wheat, are used for the production of ethanol. In thisprocess, raw starch hydrolyzing enzymes are used to breakdown the starchinto monomeric glucose which is then metabolized by the microorganism(yeast, Saccharomyces cerevisiae) to produce ethanol. This process mayalso be termed as raw starch hydrolysis or cold cook process.

Compositional Analysis of Raw Materials

Corn is first processed with a Hammer mill using 0.5 mm to 2.0 mmscreens to grind the flour to the required particle size. The percentsolids and percent moisture of the corn flour and preblend used infermentation is determined by mass loss on drying in a 100° C. oven.Preblend is defined as a nutrient source derived from recycled plantmakeup water composed of diluted and partially clarified thin stillage.The fat content of the flour is determined by accelerated fat extractionutilizing an extraction system (Dionex ASE 350) with hexane as theextracting solvent.

Yeast Propagation and Conditioning

First, 1-3 colonies of yeast isolated off yeast extract and soy peptonecontaining 3% glucose (YP medium) agar plate, or alternatively slurrieddry yeast or crème yeast, were used to inoculate 50 mL of YP culturemedia in a shake flask. This was then allowed to shake in a water bathat 150 rpm overnight for approximately 17 hours at 30° C. Theconditioning medium was then prepared in a 1 L Pyrex bottle capped witha lid with a hole to release carbon dioxide produced duringfermentation. To the fermenter bottle, corn flour was added and slurriedup to a final solids loading of 32% using preblend. The slurry was pHadjusted to 4.5 using 10% (% v/v) sulfuric acid. In addition, anappropriate amount of antibiotic, urea, a cocktail of α-amylases andglucoamylases are added to the slurry according to U.S. Pat. No.7,842,484. Yeast culture at approximately 1.0E+07 cells mL⁻¹ was addedto the fermenter to give a final number of 7.0E+08 yeast cells in thefermenter. The conditioning fermentation was allowed to ferment in awater batch shaking at 150 rpm at 30-32.2° C. for eight hours.

Fermentation

Fermentation was carried out as in the conditioning step according toU.S. Pat. No. 7,842,484 unless stated otherwise. For fermentation, a 500mL Pyrex bottles were used for a total fermentation volume of 250 mL.The corn was slurried with preblend to a total percent solids of 36.5%.In addition, an esterase such as a lipase (Novozymes Eversa Transform2.0), was added to the slurry as well. The dose of the enzyme is basedupon the total weight of corn fat present in the fermenters. A typicaldose is 0.4% (% w/w), although experiments with 0.04% and 4.0% wereperformed as well. Fermentation in the bottles was allowed to progressfor 88 hours, at which point the beer was sampled and harvested for oilanalysis.

Oil Extraction and Analysis Oil Extraction

The oil was extracted from the entire volume of beer remaining aftersampling. First, the beer was centrifuged at approximately 4° C. for 20minutes at 4500 rpm in a bench centrifuge. The resulting floating oilemulsion was then removed. The emulsion was put in 50 mL conical tubesto which approximately 10-20 mL of chloroform was added and vortexed.Then 10-20 mL of deionized water was also added to help with separation.The 50 mL tubes were then centrifuged at 3000 rpm for five minutes. Thebottom layer (chloroform+oil) was pulled off and put into tared glassvials and inserted into a turbovap to evaporate off the solvent. Theresulting dry oil was then used to quantify fatty acid ethyl esters.

Fatty Acid Ethyl Ester Determination by Gas Chromatography

Approximately 50 mg of the extracted oil was added to a 10 mL volumetricflasks to which xylene was added to the 10 mL graduation. Externalstandards including ethyl palmitoleate, ethyl oleate, and ethyllinoleate were used to generate standard curves to determine the amountof each individual fatty acid ethyl ester present in the extracted cornoil. Standard concentrations used ranged from 0.02 mg mL⁻¹ to 0.40 mgmL⁻¹. The samples and standards were run on a gas chromatograph (GC)equipped with a split/splitless injector (with splitless glass liner)and flame ionization detector (FID). Also, the GC was equipped with aPhenomenex Zebron ZB-Waxplus column (30 mL×0.32 mm ID×0.25 μm df).Analysis was conducted by injecting 1 μL of the sample into the inletheld at 250° C. The oven was initially set at 170° C. and followed anoven temperature gradient of 2° C. min⁻¹ up to 200° C. holding for 15minutes, followed by a temperature gradient of 5° C. min⁻¹ up to 230° C.holding for nine minutes. The detector was maintained at a temperatureof 250° C. Hydrogen was used as the carrier gas and the flow wascontrolled in constant flow mode at 1.80 mL min⁻¹.

Major ethyl esters in corn oil are ethyl palmitate, ethyl stearate,ethyl oleate, ethyl linoleate, and ethyl linolenate. In one embodiment,the ethyl esters include about 5% w/w to about 22% w/w ethyl palmitate,about 1% w/w to about 5% w/w ethyl stearate, about 23% w/w to about 30%w/w ethyl oleate, about 53% w/w to about 60% w/w ethyl linoleate, andabout 1% w/w to about 2% w/w ethyl linolenate of FAEE. In oneembodiment, the ethyl palmitate is about 23% w/w to about 35% w/w, ethylstearate is about 1% w/w to about 5% w/w, ethyl oleate is about 10% w/wto about 22% w/w, ethyl linoleate is about 40% w/w to about 52% w/w, andethyl linolenate is about 2% w/w to about 3% w/w of FAEE. In oneembodiment, the ethyl palmitate is about 23% w/w to about 35% w/w, ethylstearate is about 1% w/w to about 5% w/w, ethyl oleate is about 10% w/wto about 22% w/w, ethyl linoleate is about 40% w/w to about 61% w/w, andethyl linolenate is about 2% w/w to about 3% w/w of FAEE. With theaforementioned instrument parameters, ethyl palmitate would elute around11 minutes, ethyl stearate around 16.5 minutes, ethyl oleate around 17minutes, ethyl linoleate around 18 minutes, and ethyl linolenate around19.5 minutes. A standard curve of each ethyl ester is obtained to givethe slope and y-intercept for quantitation. Ethyl palmitateconcentration is determined by the ethyl palmitoleate standard curve,ethyl stearate and ethyl oleate concentration are determined by theethyl oleate standard curve, and ethyl linoleate and ethyl linolenateconcentrations are determined from the ethyl linoleate standard curve.The total FAEE content of each sample is determined using the equationbelow.

${\% \mspace{14mu} {{FAEE}( {\% \mspace{14mu} {mg}\text{/}{mg}} )}} = {\sum\frac{A_{x} - {y_{int} \cdot 10}}{S{\cdot m}}}$

Where:

-   -   A_(x)=Area corresponding to the peaks for the individual esters    -   Y_(int)=y-intercept of the linear regression    -   S=Slope of the linear regression    -   m=Mass of the sample, in milligrams

Example 2

Corn oil extracted from ethanol fermentation is mostly in the form oftriacylglyceride and is typically sold into limited markets (animalfeed, food grade or bio-diesel) due to lack of industrial utility. Inorder to increase the utility of the corn oil, an esterase can be addeddirectly to fermentation to facilitate chemical modification of the cornoil to give it unique properties, specifically by increasing the ethylester content. Increased ethyl ester content lends to lower viscositywhich is desirable in asphalt rejuvenation and performance gradecomposition. The transesterification/esterification of corntriacylglycerides/free fatty acids with ethanol produced duringfermentation can have several added benefits such as increased oilyield, increase yeast vitality due to liberation of free fatty acids andglycerol, as well as enhanced starch utilization.

Avoiding a high temperature liquefaction step of corn prior tofermentation has several benefits. One such potential benefit is thatthe corn oil extracted post fermentation has a higher concentration oflong chain ethyl esters. The high temperature liquefaction likelydestroys endogenous corn enzymes which contribute to the formation offatty acid ethyl esters (FAEE) (FIG. 6). With this knowledge, additionof exogenous esterase, e.g., a lipase, was added to the fermentation todemonstrate that ethyl ester content of extracted oil can be increasedeven further, e.g., greater than 60% (% w/w). FIG. 7 shows that the FAEEcontent of the extracted corn oil can be increased beyond 80% w/w.

Thus, in order to increase the utility of the corn oil, an esterase suchas a lipase can be added directly to the fermentation to facilitatechemical modification of the corn oil to give it unique properties,specifically by increasing the ethyl ester or FAEE content,respectively. The transesterification/esterification of corntriacylglycerides/free tally acids with ethanol produced duringfermentation can have several added benefits such as increase oil yield,increased yeast vitality due to liberation of free fatty acids andglycerol, as well as enhanced starch utilization.

Example 3

Recycled asphalt in pavement and shingles is often very stiff andviscous which can cause premature cracking due to lack of durability aswell as loss of workability in its use. In order to rejuvenate recycledasphalt by reducing the viscosity, softening, and increasing thedurability of asphalt mixtures, vegetable oils such as corn oil that areenhanced with fatty acid ethyl esters (see Example 2) can be mixed withasphalt binder or asphalt mixes containing recycled asphalt. High ethylester containing corn oil is shown herein to rejuvenate recycled asphaltin the aforementioned ways better than corn oil with a low ethyl estercontent.

Recycled asphalt increases the stiffness and makes asphalt blends proneto low temperature cracking (Mogawar et al., 2013). The use ofrejuvenator's such as waste vegetable oils, waste grease, re-refinedengine oil bottoms, crude tall oils, and aromatic oils have shownpromise to reduce stiffness and improve low temperature crackingcharacteristics (Zaumanis et al., 2014). Although corn oil is known toinherently have low viscosity properties due to the presence ofunsaturated fatty acids, as described herein, the inclusion of ethylesters or fatty acid ethyl esters reduces the viscosity even further andincreases its effectiveness as a rejuvenator.

An increase in the relative durability of the asphalt is determined bycalculating the increase in ΔT_(c) of aged asphalt after rejuvenationwith such a material. The ΔT_(c) is the difference between thecontinuous stiffness temperature and the continuous relaxationtemperature measured by the bending beam rheometer test (AASHTO T313).Asphalt binder with lower or more negative ΔT_(c) values extracted fromrecycled asphalt pavement have been shown to experience prematurecracking (Bennert et al., 2016).

FIG. 8 demonstrates that corn oil containing higher concentrations ofethyl esters leads to a lowering of the corn oil viscosity. FIG. 9 showsthat blending aged asphalt with higher inclusion of ethyl esters in cornoil leads to a desirable increase in the aged asphalt ΔT_(c) value.Higher concentrations of ethyl esters reduce the relaxation temperatureof aged asphalt binder, and hence improve the low temperatureproperties.

Example 4

Recycled asphalt in pavement (RAP) is often very stiff and viscous whichcan cause premature cracking due to lack of durability as well as lossof workability in its use. Distillers corn oil (DCO), e.g., producedwith added esterase in the fermentation, can be utilized to reduce theviscosity, improve the low temperature properties, as well as increasethe durability of the recycled asphalt for use in asphalt mixes. DCO maybe used with asphalt mixes containing 1% to 50% RAP in order to increasethe cracking resistance while not exceeding the rutting limit.Additionally, DCO can be used to modify the grade of various performancegrade (PG) asphalts in order to improve the low temperature properties.The composition of the aforementioned DCO contains greater than 18%fatty acid ethyl ester (FAEE) by weight.

Waste vegetable oils, waste grease, re-refined engine oil bottoms, crudetall oils, and aromatic oils can be used to modify the PG of asphalt,reducing the stiffness and improving the low temperature propertiesmaking their use more amenable to particular climates (Golalipour,2013).

Recycled asphalt and some PG asphalts are very viscous and stiff whichwould benefit from a rejuvenating or softening agent. DCO can lower thestiffness of the aforementioned asphalt and can improve the lowtemperature properties by making it less susceptible to cracking. Inaddition, DCO can be added to asphalt mixes containing 1%-50% RAP inorder to soften the asphalt, increase the durability of the asphalt, andimprove cracking resistance while not exceeding the ruttingspecification.

FIG. 10 shows the PG modification of a 64-22 asphalt to a 58-28 and52-34 with 4 and 7 percent inclusion of DCO, respectively. DCO can alsobe used in asphalt rejuvenation applications. FIG. 11 demonstrates theuse of DCO can increase the ΔT_(c) value of aged asphalt, which is ameasure of the durability of the asphalt. Thus, the inclusion of 4% DCOin a 50% RAP mixture can significantly increase the cracking resistanceas well as pass the specification for rutting, respectively.

DCO can also be used in asphalt rejuvenation of recycled asphalt presentin RAP and RAS. As asphalt is aged, the binder becomes oxidized andhardens decreasing the ΔT_(c) value indicating a loss of durability. Inorder to rejuvenate aged asphalt, DCO can be added to the recycledasphalt in order to increase the ΔT_(c) value. In addition, inclusion ofDCO increases the mix performance of RAP blends as observed as anincrease in both low and intermediate cracking resistance withoutcausing the mix to become susceptible to rutting. Typical inclusion ofRAP in asphalt mixes may range from 1% to 50%. Inclusion of DCO in RAPcontaining asphalt mixtures may range from 0.5% to 50%, e.g., 25%, basedupon the weight of the binder that includes the recycled asphalt ortotal weight of the asphalt. For a hot mix, RAP or RAS can berejuvenated by several different methods. DCO can be added onto the RAPor RAS stockpiles, added directly into the mix drum, or injected intothe virgin asphalt. RAP/RAS can be pretreated by spraying the streamprior to its addition to the mix drum. DCO can also be added to virginasphalt in storage tanks equipped with mixers or it can be added with anin-line static mixer downstream prior to reaching the mix drum.

Typical mix design of asphalt formulations with and without inclusion ofRAP and an exemplary corn oil composition, DCO, are shown in Table 7.

TABLE 7 Typical Asphalt Mix Design for Virgin, 50% RAP, and 50% RAP withInclusion of Corn Oil Compositions (“DCO”) 50% RAP + Volumetrics Virgin50% RAP DCO^(a) Requirement Total Binder, % 6.1 6.0 6.0 — Virgin Binder(PG 6.1 3.16 3.16 — 67-22), % Binder from RAP, % 0 2.84 2.84 — AirVoids, % 4.0 4.0 4.0 4.0 VMA^(b), % 16.8 16.7 16.5 >15.0  VFA^(c), % 7576 76 73-76 Ratio of Dust to 1.2 1.2 1.2 0.6-1.2 Asphalt ^(a)DCO isincluded at 4% based upon the total weight of the binder blend or 8%based on recycled asphalt binder (in the 50% mix) ^(b)VMA: Voids in theMineral Aggregate ^(c)VFA: Voids Filled with Asphalt

REFERENCES

-   Bennert et al., Transp. Res. Rec. J. Transp. Res, Board, 2574:1    (2016).-   Cox, Asphalt Binders Containing a Glyceride and Fatty Acid Mixture    and Methods for Making and Using Same. (2016).-   DiCosimo et al., in situ expression of lipase for enzymatic    production of alcohol esters during fermentation (2014).-   Golalipour, Investigation of the Effect of Oil Modification on    Critical Characteristics of Asphalt Binders. PhD Thesis (2013).-   Grichko, Fermentation processes and compositions (2004).-   Hughes et al., J. Assoc, Lab. Autom., 16:17 (2011).-   Lackey & James, Biodiesel cutback asphalt and asphalt emulsion.    (2004).-   Mogawer et al., Road Mater. Pavement Des., 14:193 (2013).-   Moreau et al., J. Am. Oil Chem. Soc. 88:435 (2010)-   Seidel & Haddock, Constr. Build. Mater. 53:324 (2014).-   van den Berg et al., Biotechol. Bioenq., 110:137 (2013).-   Zaumanis et al., Constr. Build. Mater., 71:538 (2014).-   Winkler et al., J. Auric. Food Chem., 55:6482 (2007).-   Winkler-Moser and Vaughn, J. Am. Oil Chem. Soc., 86:1073 (2009).

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification, thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details herein may be varied considerably without departing fromthe basic principles of the invention.

What is claimed is: 1-42. (canceled)
 43. A corn oil compositioncomprising corn oil with a fatty acid ethyl ester content greater than7% w/w based on the total weight of the corn oil composition; a freefatty acid content including at least one fatty acid selected from thegroup consisting of C16 palmitic, C18 stearic, C18-1 oleic, C18-2linoleic, and C18-3 linolenic; and an ion content of about 0.4 to about20 ppm.
 44. The corn oil composition of claim 43, wherein the free fattyacid content is no greater than 5% w/w based on the total weight of thecorn oil composition.
 45. The corn oil composition of claim 43, whereinthe ion content includes alkali metal ions, alkaline metal ions, orcombinations thereof.
 46. The corn oil composition of claim 45, whereinthe ion content include metal ions selected from lithium, sodium,magnesium, potassium, calcium, or combinations thereof.
 47. The corn oilcomposition of claim 46, wherein the corn oil composition includes about0.4 to about 10 ppm of the ion content.
 48. The corn oil composition ofclaim 43, wherein the fatty acid ethyl ester content includes about 23to about 35% w/w of ethyl palmitate and about 10 to about 22% w/w ofethyl oleate.
 49. The corn oil composition of claim 48, wherein thefatty acid ethyl ester content includes about 40 to about 61% w/w ofethyl linoleate.
 50. The corn oil composition of claim 43, wherein thefatty acid ethyl ester content includes about 5 to about 22% w/w ethylpalmitate, about 1 to about 5% w/w ethyl stearate, about 23 to about 30%w/w ethyl oleate, about 53 to about 60% w/w ethyl linoleate, and about 1to about 2% w/w ethyl linolenate.
 51. The corn oil composition of claim43, wherein the fatty acid ethyl ester content includes about 23 toabout 35% w/w ethyl palmitate, about 1 to about 5% w/w ethyl stearate,about 10 to about 22% w/w ethyl oleate, about 40 to about 61% w/w ethyllinoleate, and about 2 to about 3% w/w ethyl linolenate.
 52. The cornoil composition of claim 43, wherein the fatty acid ethyl ester contentincludes about 5 to about 35% w/w ethyl palmitate, about 1 to about 5%w/w ethyl stearate, about 10 to about 30% w/w ethyl oleate, about 40 toabout 61% w/w ethyl linoleate, and about 1 to about 3% w/w ethyllinolenate.
 53. The corn oil composition of claim 43, wherein the cornoil composition has a fatty acid ethyl ester content up to about 80%w/w.
 54. The corn oil composition of claim 43, wherein the corn oilcomposition further includes an unsaponifiables content no greater than3% w/w.
 55. The corn oil composition of claim 43, wherein the corn oilcomposition has a moisture content of no greater than 1% w/w.
 56. Thecorn oil composition of claim 55, wherein the corn oil composition has amoisture content of about 0.2% or greater.
 57. The corn oil compositionof claim 43, wherein the corn oil composition has an insoluble contentof no greater than 1.5% w/w.
 58. The corn oil composition of claim 43,wherein the corn oil is obtained from a fermentation residue of corn.59. The corn oil composition of claim 43, wherein the fatty acid ethylester content is greater than 18% w/w.
 60. The corn oil composition ofclaim 43, wherein the fatty acid ethyl ester content includes morepolyunsaturated ethyl esters than monounsaturated ethyl esters andsaturated ethyl esters.
 61. The corn oil composition of claim 43,wherein the fatty acid ethyl ester content includes about 54 to about 62weight percent of polyunsaturated ethyl esters.
 62. The corn oilcomposition of claim 43, wherein the fatty acid ethyl ester contentincludes about 2.9 to about 4.2 times more polyunsaturated ethyl estersthan monounsaturated ethyl esters.